Diisooctyl 2,2'-[(dioctylstannylene)bis(thio)]diacetate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

2 Generation Reproductive Toxicity Study - Read-across data (mixture DOTI: MOTI. ca. 80:20) (CAS No 26401-97-8 and CAS 26401-86-5)

No influence on reproduction parameters were observed at 20 and 60 ppm in F0 and F1 generation. There was a slight increase in stillbirths at 60 ppm (F1) which was suspected to be treatment-related.

Under the conditions of the study the NOEL of the test material for reproduction in the F0 and F1 generations was 20 ppm in the diet (equivalent to approximately 1.5 - 1.6 mg/kg bw/day).

Link to relevant study records

Reference

Reference 1

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 June 1992 to 17 May 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

1983

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Males 26 days and females 30 days
- Weight at study initiation: Males and Females: 61 to 74 g
- Housing: Except during the mating period (one male/one female per cage) and lactation (litters caged with their dams; solid bottom cages with bedding material) the males and females (F0-generation) were kept singly in stainless steel wire mesh bottom cages.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 50 ± 15 %
- Photoperiod: 12 hour cycles of light and dark. Light at approximately 150 lux at 1.5 m room height.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet: weekly
- Mixing: For the preparation of 10 kg batches, exactly weighed amounts of test material (200, 600 and 2000 mg) were pounded in a mortar with an adequate amount (maximally the tenfold) of standard diet; this premix was further diluted stepwise (by factors of maximally 10) to about 100 g; this mixture was transferred to a rotary blender and diluted in two further steps (homogenisation period of 10 minutes each) to the designated final concentration.
- Storage temperature of food: Each batch was divided into aliquots sufficient for 2-3 days and stored deep-frozen until use.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: Maximum of 3 weeks or until signs of mating (microscopic observation of sperm in the vaginal smear) were observed.
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of gestation
- Re-matings were not carried out at the request of the sponsor.
- After successful mating each pregnant female was caged individually except during the lactation phase when dams were housed with their litter. Bedding material was added to the cage on day 21 of gestation prior to the anticipated point of parturition.
- The morning on the day that all pups had been delivered was defined as day 1 of lactation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Analyses for homogeneity and stability in the test material-diet mixtures were performed by the sponsor. A sample of standardised diet (approx. 200 g) was examined for control purposes. The content of the prepared test diets were monitored monthly by the laboratory using total tin determination. Analyses were performed using the ICP-OES (inductive coupled plasma-optical emission spectrum) after extraction under pressure with nitric-sulphuric acid based on § 35 LMG 00.00-19.
- Five samples of approximately 100 grams each were drawn per concentration at five different locations (top, mid and bottom and from two further areas) for assessment of homogeneity of the mixture. Additional samples of 100 grams each per concentration were stored as a thin layer on sheet metal for periods of 1, 2, 3 and 7 days in the animal room for the assessment of stability: Homogeneity and stability samples were stored deep-frozen (at -20 °C or colder) until analyses were performed.
- The analytical results indicate that the test material diet mixtures were correctly prepared during the whole experimental period.
- Nearly all results were with the admissible limits of ± 10 % of the nominal value. A few deviations greater than 10 % occurred, mainly at the low concentration. This may have been caused by the higher scatter of the analytical results at the low concentration, observed outliers did not effect the validity of this study. The maximum deviation was 14.9 % regarding the 60 ppm and 200 ppm concentrations and 40.4 % regarding the 20 ppm concentration.

Duration of treatment / exposure:

- The F0-generation animals were exposed to the appropriate test diets seven days/week for a 70-day (10-week) premating treatment period. The male rats continued to be treated daily during the 21-day mating period and post-mating period until sacrifice. The female rats continued to be treated daily during mating and mated females continued to receive test diet during the ensuing gestation and lactation periods. Unmated females continued on the test diets until sacrifice.
- The F1-generation animals received the appropriate diet (dose-level of the parent animals of the F0-generation) seven days/week for a 98-day (14-week) premating period. The male rats continued to receive the appropriate test diets during the 21-day mating period and post-mating period until sacrifice. The female rats continued to be treated throughout the ensuing mating, gestation and lactation periods similar to the animals of the F0-generation.

Frequency of treatment:

Continuous in the diet

Details on study schedule:

- F1 parental animals not mated until 14 weeks after they were selected from the F1 litters.
- At weaning of each litter, two pups/sex/litter were chosen at random and became the parent animals for the F1-generation. If more than 25 F1 pups/sex/group had been chosen after the last litter was weaned, the excess number of pups was culled so that each litter was represented in the F1 parent generation by at least one pup/sex.
- Age at mating of the mated animals in the study: 17 to 20 weeks. To avoid brother/sister mating during setting-up the F1-generation, the position of the first half of the males of a group was changed within the males of the second half. Rematings were not carried out at the request of the sponsor.

Dose / conc.:

20 ppm

Dose / conc.:

60 ppm

Dose / conc.:

200 ppm

No. of animals per sex per dose:

F0 and F1 generations: 25 animals per sex per dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: the lowest concentration of 20 ppm was expected to be a no-effect-level in the rat with regard to systemic toxicity and reproductive/developmental toxicity; there is a sufficient margin compared to the currently recommended TDI-value; at the intermediate concentration of 60 ppm lymphatic organs could be affected, in particular in juvenile rats; 200 ppm was considered a threshold exposure for systemic toxi¬city in adult rats (body weight reduction; effects on the thymus).

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: behaviour and general condition were observed daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: condition of faeces was observed during daily controls.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly (always on the same day and at the same time of day)
- During pregnancy, beginning on day 0, body weight of all dams was registered weekly. During the lactation period these registrations were made on days 1, 4, 7, 14 and 21 post partum.

FOOD CONSUMPTION:
- Food consumption was checked and registered at intervals of 2 to 3 days (always at refilling of food containers and the same time of day) by weighing the residue.

WATER CONSUMPTION: Yes
- Time schedule for examinations: during daily controls.

Oestrous cyclicity (parental animals):

- The dams of both parental generations (F0 and F1) were scheduled for spontaneous delivery and daily observations during the lactation period.
The following parameters were determined:
- Mean pre-coital time in days: (vaginal smears were taken daily during the mating period to record the oestrus cycle and to determine day 0 p.c.)
- Mean duration of pregnancy in days (from day 0 of pregnancy to the last delivery)
- Delivery: day of completion (designated as day 1 p.p.) and signs of dystocia, if present

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible). Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example five males and three females) was carried out. Adjustments were not applicable for litters of less than eight pups; excess pups were killed and preserved in ethanol after external and internal examination.

PARAMETERS EXAMINED
The following parameters were examined in offspring:
- Number of pups (absolute): at birth (alive and dead), after 4 days of life before adjustment, after 4 days of life after adjustment, after 7 days of life, after 14 days of life, after 21 days of life, per dam at birth and after 21 days of life.
- Number of male and female pups: at birth, after 4 days of life (after adjustment) and after 21 days of life.
- Number of stillbirths: absolute and per dam.
- Number of runts (pups were considered as runts if their weight was less than 70 % of the mean litter weight): absolute and per dam.
- Number of pups with malformations: absolute and per dam.
- Body weight of pups: Individual weights were determined and documented, group mean values calculated based on litter mean values) on days 1, 4 (before and after adjustment), 7, 14 and 21 post partum.
- Functional tests performed on: Mid-air righting reflex, Auditory startle reflex and Pupillary reflex.
- Day of occurrence of the following morphological landmarks: Pinna detachment, ear opening, eye opening, testicular descent or vaginal opening.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after weaning of the last litter in the respective generation.
- Maternal animals: All surviving animals after weaning of the last litter in the respective generation.

GROSS NECROPSY
- The number of implantation scars on the endometrium were recorded in all parental females, uteri of apparently non-pregnant females were stained using the SALEWSKI-technique prior to fixation.
- Samples of the following tissues and organs were collected from all parental animals at necropsy and fixed in LILLIE's buffered formalin: all gross lesions; ovary, uterus, cervix, vagina; testis, epididymis, seminal vesicle with coagulation gland, prostate, mammary gland, pituitary, adrenal, thyroid, spleen, thymus, iliac lymph node.

HISTOPATHOLOGY / ORGAN WEIGHTS
- The following organ weights were recorded in all parental animals (F0 and F1): ovary, uterus, testis, pituitary, adrenal, thyroid, spleen, thymus and iliac lymph node.
- Haematoxylin-eosin stained sections of preserved organs and tissues as specified afore were examined histopathologically.
- All gross lesions as well as the complete set of preserved tissues of the control and high dose groups were examined in parent and young animals of the F0 and F1-generations.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed after weaning on day 22 of life.

GROSS NECROPSY
- Samples of the following tissues and organs were collected from one male and one female pup of each F1 and F2 litter at necropsy (day 22 p.p.) and fixed as appropriate: spleen, thymus, iliac lymph node

HISTOPATHOLOGY / ORGAN WEIGTHS
- The following organs were weighed in one male and one female pup from each Fi and F2 litter: spleen, thymus and iliac lymph node.
- Haematoxylin-eosin stained sections of preserved organs and tissues as specified afore were examined histopathologically.
- All gross lesions as well as the complete set of preserved tissues of the control and high dose groups were examined in parent and young animals of the F0 and F1-generations.

Statistics:

- For intergroup comparison, the individual data were processed as appropriate to present group mean values and standard deviations, percentages per group and/or group means of percentages per litter. In addition, reproductive indices were calculated.
- The following standard statistical tests (each one performed at a 5 % α- and 5 % β-level) were applied in order to compare dose-level group values with the respective control values:

DUNNETT-Test:
- adult body weight and body weight change; food consumption
- organ weight, body weight of the pups
- mean number of implantations and pups per dam
- mean number of viable pups per litter

Multiple Chi-Square-Test and, if necessary, pairwise FISHER's Exact:
- qualitative findings in the dams (clinical signs, necropsy findings)
- percentages of pre- and postnatal loss
- percentages of dead young rats on day 1 post partum
- sex ratio
- incidence of malformations
- percentage of anomalous pups
- percentages of inseminated and of pregnant females
- fertility index, gestation index, viability index, lactation index

KRUSKAL-WALLIS-Test:
- group mean of percentages of prenatal, perinatal and preweaning loss per dam (litter)
- group mean of percentages of anomalous pups per litter
- mean gestation length
- group mean of percentages of pups reaching criterion (functional tests and developmental landmarks) per litter
- mean number of mating days until insemination

Reproductive indices:

- Fertility index in % = (number of pregnant rats/ number of rats used) x 100
- Gestation index in % = (number of rats with living litter/ number of pregnant rats) x 100

Offspring viability indices:

- Viability index in % = (number of pups living on day 4 before adjustment/ number of living pups at birth) x 100
- Lactation index in % = (number of pups living on day 21/ number of pups living on day 4 after adjustment) x 100

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

- The lowest tested concentration (20 ppm in the diet) did not influence behaviour and external appearance.
- The male parent animals of the intermediate tested concentration (60 ppm in the diet) remained inconspicuous. A female of this group showed tonoclonic convulsions on the 13th and 14th lactation day (test week 16), which were considered to be of spontaneous nature and not substance related.
- At the highest tested dose-level (concentration: 200 ppm in the diet) a female animal showed a 14-day lasting loss of hair during the post-lactation period (test weeks 18 and 19), the right flank and the right hind limb were affected. Additionally, a slight transient dyspnoea was observed in this dam during two days of test week 19 (post-lactation period). These findings were considered to be of spontaneous nature and not treatment related.
- The faeces of the test material treated animals did not show any differences from those of the control group with reference to colour and consistency.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

- At the highest tested dose-level (200 ppm in the diet) one male was found dead in the morning of the first day of test week 24 (post-mating phase). It died unobserved during the night and was regarded a spontaneous event.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

- Body weight and body weight gain were within the range of the controls during all phases of the experiment (males: pre-mating, mating, post-mating; females: pre-mating, mating, gestation, lactation and afterwards until dissection) at all tested concentrations (20 ppm, 60 ppm and 200 ppm in the diet).
- In the low-dosed female animals (20 ppm) body weight and body weight gain did not differ from that of the controls during pre-mating, mating, gestation, lactation and afterwards until dissection.
- At 60 and 200 ppm in the diet slightly increased maternal body weights were registered during gestation and lactation. These differences may be due to the higher maternal body weight values at start of the gestation period. As a clear dose-relationship is lacking these results are regarded as spontaneous.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- None of the tested concentrations (20 ppm, 60 ppm and 200 ppm in the diet) caused any impairment of the food consumption in the male parent animals compared with the control group. All values were within the control range during the pre-mating and post-mating phase. Several very slight and significant (at p ≤ 0.05 or p ≤ 0.01) differences from the control group belong within the spontaneous range.
- This was generally also true for the female parent animals of all groups - also during the gestation period. However, during the lactation period of the dams treated with a concentration of 200 ppm in the diet the values of the absolute food consumption were below those of the control group (between minus 6 % (days 7 to 14) and minus 9 % (days 14 to 21; not significant at p ≤ 0.05)).

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

Drinking water consumption did not show any test material-related changes.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- Histopathological examination revealed an increased incidence of thymic involution (significant at p ≤ 0.01 in the male animals of the 200 ppm dose) due to an expected well-known effect of the compounds in the test material.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

- Eight female parent animals across all concentrations did not become pregnant though sperm was found in the vaginal smear.
- One control female animal was not mated during the whole mating period. At necropsy of this animal, no ovaries were found during macroscopic examination, however, the tubes seemed to be normally developed.
- 20 ppm and 60 ppm concentrations did not influence the reproduction parameters of rats. Indices of mating, fertility, gestation and the pregnancy rates were within the range of the control group. The mean pre-coital time, duration of pregnancy in days and duration of parturition in hours did not show any substance-related influence. All dams remained inconspicuous during the delivery. The number of pups born was not affected; birth body weight of the pups lay within the range of the control.
- Treatment with the highest concentration (200 ppm in the diet) led to a slightly decreased fertility index of 88.0 % (3 of 25 females did not become pregnant, control: 100.0 %, fertility index). The difference was not statistically significant. The fertility index in this group was within the range observed in historical control animals from previous studies in our laboratory. Therefore, the above-mentioned difference is considered as a chance finding.
- The mean body weight of the pups at birth was very slightly reduced (male pups: by approx. 3 %, female pups: by approx. 4 %). These results did not differ significantly from those of the control group (at p ≤ 0.01).
- Eleven stillbirths were found in the treated groups: 5 at 20 ppm, 2 at 60 ppm and 4 at 200 ppm (control: 6 still births). These findings and 3 runts (2 at 20 ppm, 1 at 60 ppm) belong to the spontaneous range (control: 1 runt).
- One pup of the low and one of the high concentration were malformed. Both showed stump-tail, shifted and fused dorsal, lumbar and coccygeal vertebrae, the pup from the high dose in addition crossed-legs and anal atresia. These malformations were spontaneous with reference to their type and number.

Key result

Dose descriptor:

NOEL

Effect level:

20 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

Remarks on result:

other: Approximately 1.5 mg/kg bw/day

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

F1 pups/ young animals until weaning
- Sex distribution of pups from parents treated with concentrations of 20 ppm and 60 ppm in the diet did not show any test material-related influence until weaning (at the end of the 21-day lactation period). The functional test in young animals (mid-air righting, auditory startle and pupillary reflex) revealed no test material-related influence. There was no delay of morphological landmarks (pinna detachment, ear and eye opening, testicular descent and vaginal opening).
- At 200 ppm sex distribution of pups was not influenced. The functional tests in young animals revealed no substance-related changes, observations of morphological landmarks showed a slight but significant (p ≤ 0.05) delay of vaginal opening (mean days in the treated group: 35.4, in the control group: 33.2).

F1 Parental generation
- The lowest tested concentration (20 ppm in the diet) did not influence behaviour and external appearance of the male parent animals. One of the female parent animals showed opisthotonus on test day 77 and transient (tono-)clone convulsions, accompanied by vocalisation on test days 77 and/or 91 and 98. In addition, excitement was observed in this animal on test days 77 and 98.
- The female parent animals of the intermediate tested concentration (60 ppm in the diet) remained inconspicuous. The male animal no. 109-1 of this group showed a haemorrhagic snout from the 104th to 109th test day. Male rat no. 122-1 had to be sacrificed prematurely on the 94th test day. Prior to death dyspnoea, a haemorrhagic snout and pilo-erection were observed from test day 85 onwards and reduced motility as well as haemorrhagic canthi from test day 90 onwards. In addition, sedation was seen on test day 91, ataxia from test day 91 onwards.
- At the highest tested dose-level (200 ppm in the diet) the male parent animal no. 152-1 showed a paralysis of the hind extremities from test day 8 onwards, this rat had to be sacrificed prematurely on test day 9. Two of the 25 female parent animals died prematurely. One of them on the 123rd test day at littering unobserved during the night (rat no. 187-1). The second (dam no. 198-1) died on the 129th test day. Prior to death, pale eyes were observed in this dam on the 1st day after the start of littering (126th test day), delivery was not yet completed. Pilo-erection was observed in this animal from the 128th test day until its death.
- The changes observed are considered to be spontaneous events and not substance-related. There were no histopathological correlates to confirm a test material-related effect.
- The faeces of the test material-treated animals did not show differences from those of the control group with reference to colour and consistency.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

F1 pups/ young animals until weaning
- Viability and lactation indices were similar to those of the control group at 20 and 60 ppm.
- The viability index at 200 ppm was within the control range. Although the lactation index was significantly (at p ≤ 0.05) decreased at 200 ppm this was regarded as a spontaneous finding since the mean value remained in the range of historical controls (86.3 to 100 %). Furthermore it should be taken into account that this difference was mainly due to the almost complete loss of the litter of two females (only one surviving pup each) in group 4.

F1 Parental generation
- All male and female parent animals at 20 ppm survived until termination of the study.
- At 60 ppm one male had to be sacrificied prematurely on day 94.
- At 200 ppm one male had to be sacrificied prematurely on day 9. Two females died during the study. Deaths were deemed not to be test-material related since no histopathological changes were seen to correlate with the deaths.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

F1 pups/ young animals until weaning
- Body weight of pups from parents treated with concentrations of 20 ppm and 60 ppm in the diet did not show any substance-related influence until weaning (at the end of the 21-day lactation period).
- Body weight of pups from parent animals treated with a concentration of 200 ppm in the diet was below that of the control group from the time-point of birth until weaning (male pups: between approx. 3 and 19 %; female pups: between approx. 4 and 21 %. Significances (at ≤ p 0.01) were calculated for both sexes after 14 and 21 lactation days.

F1 Parental generation
- Body weight and body weight gain were within the range of the controls during all phases of the experiment (males: pre-mating, mating, post-mating; females: pre-mating, mating, gestation, lactation and afterwards until dissection) at the two lower tested concentrations (20 ppm, 60 ppm).
- Body weight of the male rats treated with a concentration of 200 ppm was significantly (at p ≤ 0.01) below that of the control group from test week 1 during the whole pre- and post-mating phase (at p ≤ 0.05 in test week 1, at p ≤ 0.01 from test week 2 onwards). Body weight and body weight gain of the high-dosed female animals were within the range of the controls.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

F1 Parental generation
- None of the tested concentrations (20 ppm, 60 ppm and 200 ppm in the diet) caused any impairment of the food consumption in the male parent animals compared with the control group. All values were within the control range during the pre-mating and post-mating phase. Several very slight and significant (at p ≤ 0.05 or p ≤ 0.01) differences from the control group belong within the spontaneous range.
- This was generally also true for the female parent animals of all groups - also during the gestation period. However, during the lactation period of the dams treated with a concentration of 200 ppm in the diet the values of the absolute food consumption were below those of the control group between minus 12 % (days 1 to 7), minus 15 % (days 7 to 14) and minus 17 % (days 14 to 21; significant at p ≤ 0.05).

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

F1 Parental Generation
- Drinking water consumption did not show any test material-related changes.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

F1 pups/ young animals until weaning
- Generally, the relative organ weights determined from one young animal/sex/litter/group (spleen, thymus, iliac lymph node) at 20 and 60 ppm were within the control range. However, the relative thymus weight showed a slight tendency towards decrease in male and female rats at 60 ppm (in the females statistically significant at p ≤ 0.05).
- Relative weights of the thymus of the F1-pups at 200 ppm were significantly (at p ≤ 0.01) below those of the control group in both sexes. The relative spleen weight was reduced in male and female young rats, also, a significant (at p ≤ 0.01) reduction was found for the females.

F1 Parental Generation
- Starting at 60 ppm a slightly reduced relative thymus weight was observed for the males. At the high concentration (200 ppm) the relative thymus weight was significantly below the control in both sexes (males at p ≤ 0.05, females at p ≤ 0.01). Otherwise the relative organ weights did not show any treatment-related influence at the tested concentrations of 20 ppm, 60 ppm and 200 ppm in the diet. The slight but statistically significant increases in relative adrenal weights of the female animals at 20 ppm and in pituitary weight of the male animals at 200 ppm are considered as incidental as these differences were within the range observed in historical control animals.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

F1 pups/ young animals until weaning
- The macroscopic inspection at necropsy of 20 and 60 ppm groups revealed no test material-related pathological findings.
- At 200 ppm necropsy generally revealed no test material-related changes, however, the last surviving pup of one female was found moribund at termination of the lactation period and had to be sacrificed. It showed emaciation, tremor, thin fur and an empty gastro-intestinal tract.

F1 Parental Generation
- There were, no substance-related macroscopically visible observations at necropsy in any of the surviving male and female parent animals treated with concentrations of 20 ppm and 60 ppm in the diet. A few slight changes in individual animals belong to the spontaneous range.
- The prematurely sacrificed male animal at 60 ppm showed a severely enlarged pituitary; the organ nearly covered the whole base of the skull; the pituitary was soft and glassy, it was left at its place. This change was considered a spontaneous event and hence not treatment-related.
- At 200 ppm there were no treatment-related macroscopically visible observations at necropsy in any of the surviving male and female parent animals. A few slight changes in individual animals belong to the spontaneous range and were not treatment-related. The implantation count revealed no treatment-related changes at all tested concentrations.
- The prematurely sacrificed male rat and the prematurely deceased female rats showed the following pathological findings at necropsy:
No. 152-1 male: paralysis of the hind extremities; brain: subdural haemorrhages in the region of the roof and base of the skull; a few blackish foci in the lungs (0 approx. 1 mm); a reddened and haemorrhagic urinary bladder; multiple haemorrhagic foci and several ulcers in the gastric wall (0 1-2 mm); rudimentary and haemorrhagic seminal vesicle.
No. 187-1 female: no pathological findings; 6 foetuses and 6 placentae in the uterus
No. 198-1 female: liver and kidneys pale (anaemia); two whitish-beige foci in the liver (0 8mm each)
- These changes were considered as spontaneous events and hence not substance-related.

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

F1 pups/ young animals until weaning
- No histopathological changes of spleen, thymus or iliac lymph node were noted in the pups of the F0-generation at any concentration.

F1 Parental Generation
- Histopathology of the prematurely sacrificed male rat revealed mainly a foreign body granuloma in the lungs and haemorrhages in the seminal vesicles in the urinary bladder.
- Histopathology revealed a lympho-histiocytic infiltration in the uterus in one female and a multicellnecrosis in the liver of the other female. These changes were considered as spontaneous events and hence not substance-related
- Histopathological examination of all other animals revealed an increased incidence of thymic involution (significant at p ≤ 0.01 in the male animals of the 200 ppm dose) due to an expected well-known effect of the compounds in the test material.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

F1 Parental Generation
- In total, thirteen female parent animals did not become pregnant though sperm was found in the vaginal smear: four in the control group, one at the 20 ppm concentration, two at the 60 ppm concentration and three at the 200 ppm concentration in the diet. The number of treated female animals which did not become pregnant though sperm was found in the vaginal smear was not higher than the number of control animals which did not become pregnant.
- Except for one male parent animal at the lowest tested concentration (20 ppm) which showed rudimentary testicles and epididymides at necropsy and which did not mate its female partner, the 20 ppm and 60 ppm concentrations (in the diet) did not influence the reproduction parameters of rats. Indices of mating, fertility, gestation and the pregnancy rates were within the range of the control group. The mean pre-coital time, duration of pregnancy in days and duration of parturition in hours did not show any substance-related influence. All dams remained inconspicuous during the delivery. The number of pups born was not affected, birth body weight of the pups lay within the range of the control.
- The gestation index of the animals treated with the highest concentration (200 ppm in the diet) was slightly decreased to 95.2 % (control: 100.0 %). This finding was connected with the delivery results of dams no. 187-1 and 198-1 and is considered to be without any test material-related influence.
- One animal died during littering, it delivered 13 stillbirths, a further 6 foetuses were found to be still in its uterus. In another female the duration of parturition was prolonged, delivery lasted for more than 48 hours. All pups were stillbirths. Another died on the nominal 2nd lactation day. The external development of the foetuses and pups from these two female parent animals was inconspicuous. In addition another of this group showed a prolonged parturition, delivery lasted for more than 24 hours. Living pups and stillbirths were found in this animal. Altogether, the mean duration of parturition in hours was moderately increased compared with the control group. The dams treated with 200 ppm needed an average of 12.4 hours for delivery (control: average of 10.5 hours).
- Normal values were recorded for the mean pre-coital time in days, mating and fertility indices, pregnancy rate and mean duration of pregnancy in days.
- The following number of stillbirths were found in the treated groups: 5 at 20 ppm, 15 at 60 ppm and 26 at 200 ppm (control: 6 stillbirths). The result of the lowest tested concentration of 20 ppm was within the control range, at 60 ppm the number of stillbirths was slightly increased though the group 3 also contained more litters with a pup number above 15 than the control group. Hence the increase in stillbirths in group 3 is considered as a suspected test material-related effect, the effect at 200 ppm was considered clearly test material-related (high dose: significant at p ≤ 0.05). There were 4 spontaneous runts, 3 at 20 ppm and 1 at 60 ppm. The incidence lies within the spontaneous range. None of the pups of the low-, intermediate- and of the high-dosed group were malformed.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

The functional test in young animals (mid-air righting, auditory startle and pupillary reflex) revealed no test material-related influence.

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

20 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

Remarks on result:

other: Approximately 1.6 mg/kg bw/day

Clinical signs:

no effects observed

Description (incidence and severity):

- Sex distribution of pups of the F1-generation treated at all concentrations in the diet did not show any treatment-related influence until weaning (at the end of the 21-day lactation period).
- At 20 and 60 ppm there was no delay of morphological landmarks (pinna detachment, ear and eye opening).
- Observations of morphological landmarks at 200 ppm showed no treatment-related changes, except a slight delay (pinna unfolding, eye and ear opening).

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

- The viability and lactation indices at 20 and 60 ppm were similar to those in the control group or within the background data.
- The viability index was markedly decreased at 200 ppm (high-dosed group: 82.0 %, control group: 95.7 %).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Body weight of pups of the F1-generation treated at concentrations of 20 and 60 ppm in the diet did not show any treatment-related influence until weaning (at the end of the 21-day lactation period).
- At 200 ppm the body weight of pups from parent animals was below that of the control group from the time-point of birth until weaning (between approx. 3 and 19%). Significances (at p ≤ 0.01) were calculated after 4 lactation days in both sexes and after 7, 14 and 21 lactation days in the female pups.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

- At all concentrations the organ weights determined from one young animal/sex/litter/group (spleen, thymus, iliac lymph node) were within the range of the control groups.

Gross pathological findings:

no effects observed

Description (incidence and severity):

- At all concentrations the macroscopic inspection at necropsy revealed no treatment-related pathological findings

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

- At 20 and 60 ppm the functional tests in young animals (mid-air righting, auditory startle and pupillary reflex) revealed no treatment-related influence.
- The functional tests in young animals from the 200 ppm group (mid-air-righting, auditory startle and pupillary reflexes) revealed no treatment-related changes, except a slight increase of negative findings was found for the auditory startle reflex.

Developmental immunotoxicity:

not examined

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

200 ppm

Treatment related:

not specified

Relation to other toxic effects:

not specified

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

No influence on reproduction parameters were observed at 20 and 60 ppm in F0 and F1 generation. There was a slight increase in stillbirths at 60 ppm (F1) which was suspected to be treatment-related.
Under the conditions of the study the NOEL of the test material for reproduction in the F0 and F1 generations was 20 ppm in the diet (equivalent to approximately 1.5 - 1.6 mg/kg bw/day).

Executive summary:

The reproductive toxicity of the test material was investigated in a two-generation reproduction toxicity test conducted in accordance with the standardised guideline OECD 416 under GLP conditions using rats.

The test material was administered to the animals through their diet which was available to them ad. libitum at 20, 60 or 200 ppm. The concentrations of the doses were analytically verified. 25 animals per sex per dose were treated at each concentration.

The F0-generation animals were exposed to the appropriate test diets seven days/week for a 70-day (10-week) premating treatment period. The male and female rats continued to be treated daily during the 21-day mating period and the females continued to be treated during the ensuing gestation and lactation periods. The F1-generation animals received the appropriate diet (dose-level of the parent animals of the F0-generation) seven days/week for a 98-day (14-week) premating period. The male and female rats continued to receive the appropriate test diets during the 21-day mating period and females for the gestation and lactation periods similar to the animals of the F0-generation.

The no-observed-effect-level (NOEL) for the males and females of the F0-generation was 20 ppm (approx. 1.5 mg/kg bw/day). Starting at 60 ppm (approx. 4.4 mg/kg bw/day) a slightly reduced relative thymus weight for the males was observed. At 200 ppm (approx. 14.7 mg/kg bw/day) the incidence of thymic involution was increased. The changes of the thymus represent an expected well-known effect of compounds in the test material.

The no-observed-effect level (NOEL) for the F1-generation until weaning was 20 ppm (approx. 1.6 mg/kg bw/day). At 60 ppm (approx. 4.7 mg/kg bw/day) the relative thymus weight was decreased in male (slightly) and female pups (significantly). At 200 ppm (approx. 15.9 mg/kg bw/day) the relative thymus weight was significantly reduced in both sexes. The body weight of the pups at birth was slightly reduced and the body weight gain was distinctly inhibited. For the F1-generation post lactation, the no effect dose-level was also 20 ppm (approx. 1.6 mg/kg bw/day). Starting at 60 ppm (approx. 4.7 mg/kg bw/day) a slightly reduced relative thymus weight was observed for the males. In addition, a suspected increase in still births was observed. 200 ppm (approx. 15.9 mg/kg bw/day) caused moderate toxicity such as reduced body weight and reduced food consumption and influenced reproduction parameters (increased number of stillbirths, decreased viability index, decrease in pup weights). The relative thymus weight of males and females was significantly below the controls and the incidence of thymic involution was increased in the male animals.

In all treated groups no effects on organ weights nor histopathological changes were observed in the F2-generation. No teratogenic effect was observed.

No influence on reproduction parameters were observed at 20 and 60 ppm in F0 and F1 generation. There was a slight increase in stillbirths at 60 ppm (F1) which was suspected to be treatment-related.

Under the conditions of the study the NOEL of the test material for reproduction in the F0 and F1 generations was 20 ppm in the diet (equivalent to approximately 1.5 - 1.6 mg/kg bw/day).

Reference 2

Endpoint:

three-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Not specified

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

The reproductive toxicity of the test material was investigated in a three generation reproductive study, performed on male and female Long Evans rats using a non-standard method.
Animals were treated with the test material in their diet at 20, 50 and 100 ppm, they were mated on the 79th day of treatment when they were at least 100 days old. Each female was exposed to two different males during a two week mating period, all pups from the first litter born were discarded at weaning and the parent animals were mated again ten days later. The protocol was followed in this way for three generations. Litters greater than 10 in number were reduced to ten on the fifth day by selecting runts, where possible, to discard. On day 21 the weanlings were counted and weighed and either sacrificed or saved for continuation on the diets.
A statistical analysis was made of numbers of pups born in each litter, precent survival in each group, weights of pups at 21 days, weights of parent rats at time of sacrifice and organ/body weight ratios of F3b weanlings (brain, liver, kidneys).

GLP compliance:

no

Remarks:

This study pre-dates the inception of GLP

Limit test:

no

Species:

rat

Strain:

Long-Evans

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Approximately 21 days
- Housing: Males were housed in groups of 5 and females were housed individually
- Diet: ad libitum
- Water: ad libitum

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- The test material was given to animals mixed into their diet (pelleted food) ad. libitum

Details on mating procedure:

- M/F ratio per cage: 1:1. Males were always placed into the females cage.
- Animals were mated on the 79th day of treatment when they were at least 100 days old.
- Length of cohabitation: Each female was exposed to two different males during a two week period
- All pups from the first litter born were discarded at weaning and the parent animals were mated again ten days later.
- This protocol was followed for three generations.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

Animals were treated for 100 days before mating and then until litters were born and they were no longer needed. The protocol was followed in this way for three generations.

Frequency of treatment:

Continuous in the diet

Details on study schedule:

- Litters greater than 10 in number were reduced to ten on the fifth day by selecting runts, where possible, to discard. On day 21 the weanlings were counted and weighed and either sacrificed or saved for continuation on the diets.
- Age at mating of the mated animals in the study: At least 100 days old

Dose / conc.:

20 ppm

Dose / conc.:

50 ppm

Dose / conc.:

100 ppm

No. of animals per sex per dose:

10 males and 20 females per dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Animals received the same levels of test material doses in their diet as the animals in a 2 year feeding study received.

Parental animals: Observations and examinations:

- General observations were made of the animals
- Parental animals were weighed on the day of sacrifice.

Litter observations:

- The number of pups in every litter was counted on the day of birth and the fifth day. Litters greater than ten in number were reduced to ten on the fifth day by selecting runts where possible for discard. On the 21st day the weanlings were counted and weighed and either sacrificed or saved for continuation on the diets.

Postmortem examinations (parental animals):

- Parent rats were weighed sacrificed and examined grossly when they were no longer needed.

Postmortem examinations (offspring):

- When the F3b litters were 21 days old ten male and ten female pups from each group were selected at random for autopsy. Individual body weights and brain, liver and kidney weights were recorded.
- Sections of brain, heart, lung, liver, spleen, kidney and testes were preserved in formalin for histological examination.

Statistics:

A statistical analysis was made of numbers of pups born in each litter, percentage survival in each group, weights of pups at 21 days, weights of parent rats at time of sacrifice and organ/ body weight ratios of F3b weanlings. The Dunnett test was utilised for determining significant deviation of treated groups from control group values. Two sets of data, number of litters produced and number of litters where all pups died were analysed by the Chi square test.

Clinical signs:

no effects observed

Description (incidence and severity):

- The appearance and behaviour of the surviving rats in the experimental groups were indistinguishable from those of the control group in all generations. Thinning or absence of hair on the head, flanks or bellies occurred occasionally in most groups, including the control group.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

- There were no deviations in parent weights from control values in either males or females in any experimental group.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

- The number of litters produced in each group by F0 females at each of two matings was better than the expected frequency for this animal strain.

Clinical signs:

no effects observed

Description (incidence and severity):

-The appearance and behaviour of the surviving rats in the experimental groups were indistinguishable from those of the control group in all generations. Thinning or absence of hair on the head, flanks or bellies occurred occasionally in most groups, including the control group.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

- An effect on pregnant or nursing mothers was seen in several of the groups.
- Four of the F1b females died in the high test group, three before birth and one after. Three females died in the middle dose group whilst pregnant. At the second mating of F1b females two died in the high dose group and two died in the middle dose group.
- After the first mating of the F2b females one mother and litter died in the high dose group. After the second mating of the F2b females one mother and litter died in the high dose group and one mother and litter in the middle dose group died.
- An F2b adult male from the high dose group died during the study after mating.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

- There were no deviations in parent weights from control values in either males or females in any experimental group.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- Gross examination of sacrificed animals revealed no abnormalities other than isolated kidney lesions and occasional pathology of other organs which - could not be related to treatment.
- Gross examination of the F2b adult male from the high dose group that died during the study after mating, showed that the animal developed a large subcutaneous tumour.
- In three of the females that died during pregnancy or nursing lung pathology was noted, two animals had mottling over the surface of the lobes and one had bright orange lungs. The possibility of artefacts in these animals could not be ruled out.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

- The middle dose F1b females produced significantly fewer litters in the first mating than the control group (10 compared with 18).
- Among the F2b females three groups produced significantly fewer litters. In the middle dose group 20 females produced 4 and 5 litters in two matings, in the low group 20 females produced 7 and 12 litters and in the high dose group 19 females produced 5 and 9 litters. This is compared with the 20 control females who produced 19 litters each time. The same females were not necessarily barren in both breedings.
- As a result of the low incidence of litter production observed some of the F2b females which did not produce litters at the second mating were held for ovulation and fertility testing.
- The average number of pups born per litter in all groups varied from 9.1 to 12.2 in the first generation, from 8.5 to 10.6 in the second generation and from 8.7 to 11.1 in the third generation. None of the treatment groups deviated significantly from the control groups.

Clinical signs:

no effects observed

Description (incidence and severity):

- The appearance and behaviour of the surviving rats in the experimental groups were indistinguishable from those of the control group in all generations. Thinning or absence of hair on the head, flanks or bellies occurred occasionally in most groups, including the control group.

Dermal irritation (if dermal study):

no effects observed

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

- The survival of pups in the F1 generation was comparable for all groups except for the high dose of the test material, where % survival was significantly less than the control group litters (77 % compared to 97 %. The middle dose of test material was also low, 78 %, but this value was not significant at 0.05 probability level. However both of these results are within the expected range for the strain and the significant value is seen as a result of the unusually high survival rate in the control group.
- In the F2 generation the only group with a significantly lower survival rate than the control was the middle dose of test material, where 63 % survived weaning compared to 93 % in the control group.
- The high mortality rates in some of the groups was mainly caused by all pups dying in a few litters, the chi-squared test revealed no significant difference in the frequency of total litter mortality.
- In addition to the mortality occurring between birth and weaning, a substantial number of pups which had been saved for continuation on the diets died shortly after weaning (between 22 and 29 days of age). Deaths occurred after a brief interval of prostration and no abnormalities were noted upon necropsy. These post-weaning deaths were first seen in F1b litters but increased in frequency in F2b litters. All groups were affected but the middle and high dose groups were affected the most. As a result special tests were carried out on F3a and unused F3b pups at weaning.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

- The average weights of F1a and F1 b weanlings ranged from 34.5 to 40.8 g. The average weights of F2a and F2b weanlings ranged from 35.0 to 43.3 g. There was no difference in the weights of the weanlings in any groups.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Clinical signs:

no effects observed

Description (incidence and severity):

- The appearance and behaviour of the surviving rats in the experimental groups were indistinguishable from those of the control group in all generations. Thinning or absence of hair on the head, flanks or bellies occurred occasionally in most groups, including the control group.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

- In the F3 generation the survival rate of offspring was comparable in all groups at weaning.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

- The average weights of F3a and F3b weanlings ranged from 34.1 to 41.1 g. There was no difference in the weights of the weanlings in any groups.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

- The liver and kidney ratios of the males and females in all experimental groups were comparable to the control group values.
- The brain ratios in the middle and high dose groups of males were significantly less than the control group ratio. The reduced values in these two groups were probably seen because the weanlings which were selected for autopsy had somewhat higher average body weights than the controls. The average brain weights themselves for the control group and middle and high dose groups were almost identical.

Gross pathological findings:

no effects observed

Description (incidence and severity):

- Gross inspection of the weanling tissues revealed no abnormalities.

Histopathological findings:

no effects observed

Description (incidence and severity):

- All tissues of F3b weanlings submitted for histological examination were normal for males and females in all groups.

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

SUPPLEMENTARY STUDY AND DISCUSSION

> In order to investigate the post-weaning mortality pattern, which first became apparent among F1b litters and continued in F2b litters, a cross-feeding experiment was set up using all F3a pups which appeared healthy at weaning. These pups were distributed among eleven groups, using 9 to 20 males and 15 to 20 females per group.

> Weanlings were transferred to the new diets when 21 days old and were maintained on them for four weeks. Approximate food consumption was measured for the duration and rats were weighed weekly. Cages were checked daily for dead pups and at the end of four weeks the percent mortality was calculated for each group.

- There was no difference in food consumption or weight gain.

- At least 80 % of the animals in each group survived. All deaths occurred within the first week post-weaning (i.e. between the 22nd and 29th days of age). It is clear that the deaths were randomly distributed without regard to group or sex. The highest mortality occurred in a control group.

- It had been postulated earlier that the post-weaning deaths occurred as the result of the sudden change from a milk diet to a diet high in the test material. This assumption was proved false by the low mortality in two groups that went from control diet to high test material diets.

- The animals were all housed as litters, 2 to 10 in a cage, and it was noted that where deaths occurred, usually more then one pup in a litter was involved, and sometimes all the pups in a single cage died. This pattern of mortality strongly suggests the presence of an extraneous infection.

- After the final mating all F3b weanlings not selected for histological examination were retained in their original feeding groups for observation and determination of percent mortality. Once again all deaths occurred within the first week after weaning, although the pups were kept under observation for four weeks. In the control group only 2 or 3 deaths occurred in 1 litter, in the low group 7 deaths occurred involving 3 litters, in the middle group 3 deaths occurred involving 2 litters and in the high group 1 death occurred in 1 litter. It must be concluded that here again, deaths occurred in a random fashion and probably resulted from an infection. The assumption of an infection in the colony is strengthened by the fact that mortalities occurred only among rats presumably in a state of lowered resistance, that is, among pregnant or lactating females and young rats immediately after weaning.

- In an attempt to determine fertility in F2b females, the animals in the low and high groups which did not produce litters in the second mating, were held for ovulation tests. In many cases, these females had not produced litters in the first mating either. A number of normal F2b controls were also held. A vaginal smear was taken from each rat in five successive days and checked microscopically for evidence of an oestrus cycle which would indicated ovulation and fertility. 1 female in the low group had a vaginal tumour which was undoubtedly the cause of the barrenness. 1 female in the high group showed no evidence of ovulation. All remaining rats went through an oestrus cycle and were thus presumed to be fertile. As an additional test, 5 females from each group plus the control group were all mated with normal (control) adult males. One female from the middle group which had been barren in two matings was also included. All groups did equally well, considering it was the first litter for most of the experimental females and the third litter for the control females. At least 4 out of 5 females in each group produced litters (and the single female from the middle dose group produced a litter). This demonstration of fertility in the experimental females mated with normal males suggests that it was the F2b males which were responsible for the low incidence of births. A review of the individual matings of the F2b animals showed that only a small proportion of males had been effective in fathering litters. In the low dose group 4 out of 10 males fathered 7 F3a litters and 6 males fathered 12 F3b litters. In the high dose group, 4 out of 10 males fathered 5 F3a litters and 5 males fathered 9 F3b litters. The same males were probably effective in both matings but this could not be determined for sure. Considering the high rate of mortality among F2b weanlings reserved for subsequent breeding and the possibility of an infection present in the colony, it was thought that some of the surviving males had experienced sub-lethal attacks of an infection which had, however, left them sterile. If this were the case, the females which had not had litters on either trial, but which were later shown to be ovulating, had probably been paired with infertile males. On the other hand, some males, known to be fertile were unsuccessful in fathering litters when rotated to other females which were later shown to be fertile. The reasons for this are unknown. Apparently a number of factors were involved in producing this peculiar pattern of litter production.

SUMMARY

Interpretation of the reproductive studies was rendered difficult because of several changes in the mortality pattern. This first occurred among pregnant and nursing females and 3 to 4 weeks old weanlings in the groups receiving the two highest doses of test material. However, unexpected mortality in the later matings occurred as well in the control group. If an extraneous infection was in fact present in the colony, it is possible that the rats receiving the higher dose levels of test material would have been the first to succumb, due to the extra stress. On this basis, 50 ppm of the test material might be considered to be the test material effect level in that possibly resistance to infection was lowered in both mothers and weanlings. The low production of litters in several of the groups in later generations was attributed to sterility in some of the males which also may have resulted from the same infection. There were no differences among the groups in the adult weights, weanling weights, organ/ body weight ratios in weanlings or pathology.

ADDENUM STUDY

-SUMMARY

Continuation of the reproduction study further confirmed the difficulty encountered in the F2 and F3 generations was attributable to male rats. Litter production by the F3b rats was below normal in all test groups at the first mating, but the majority of females which failed to produce F4a litters were successful in producing F4b litters when mated with normal males. Most males which had been ineffective in producing F4a litters were ineffective also when mated with normal females. Microscopic examination of the testes of all F3b males revealed degenerative pathology in those males known to be non-fertile. The etiology of this degenerative change is not clear but it does not appear to be treatment related.

The addition of Brewers yeast to the diets had no significant effect on fecundity of the F3b rats or survival of their litters: litter production (F4c’s) was not improved over that seen prior to receiving the fortified diets and mortality was actually higher among F4c’s than among F4a’s. It should be noted that the F3b groups had been on the pelletised non-fortified diet until just prior to breeding for F4c litters and the degenerative pathology was apparently already present. Only the F4c groups were entirely on a fortified powdered diet. Among the F4c rats used for subsequent breeding, however, there was no evidence of reduced fertility or increased mortality in any of the groups, and the F5a and F5b litters were all within normal limits.

In conclusion the continued study of the infertility pattern noted in the earlier portion of the reproduction study revealed a reversal in the trend, with normal fecundity achieved by the fifth generation. There was correlation between testicular pathology and lack of fertility in F3b males.

-STUDY DETAILS

- F3b rats from the control, middle and high dose groups were mated when 100 days old to obtain F4a litters which were observed and discarded when 21 days old. Litter production in all groups except the control was below normal: about 65 % in both middle and high dose groups. All groups were normal however in size of litters, weights of weanlings and percent survival.

- Approximately ten days after weaning of the F4a litters, the F3b females in all groups were mated to a group of normal males which had been receiving control food but were not of the original control group. With only one exception (a control female in 831 low) all females which had failed to produce an F4a litter did produce a litter after mating with normal males (one female in the high dose group did not produce and F4a litter died before the second mating). The numbers, weights and percent survival of the F4b litters were within normal limits although the mortality among pups in all dose groups was somewhat higher than expected.

- After weaning of the F4b litters, the odd numbered F3b control female (known to be productive) were mated with those F3b males which failed to father litters in two separate attempts. Each control female was exposed to one male only. After 10 days, vaginal smears were examined daily to establish pregnancy and these data were correlated with litter production. Of the nine males tested, only 3 were successful in fathering litters; one of these was a male which had previously fathered one litter in two attempts.

- In order to rule out the possibility of the males being ineffective due to too frequent breeding, two normal males were exposed to four control females each, a week apart. Since one male fathered four litters in four attempts and the other fathered three litters in four attempts, the frequency of mating does not appear to be a factor in male ineffectiveness.

- Following the foregoing tests, all F3b rats were switched to powdered diets containing 1 % Brewers’ yeast in addition to the usual concentration of test material. The Brewers’ yeast was added to compensate for any vitamin loss which may have occurred in the pelletised food as a result of high temperatures during production.

- The fortified powdered diets were used throughout the remainder of the study. Sixteen to thirty days following the addition of Brewers' yeast to the diets, the F3b rats were mated to produce an F4c generation which was maintained for further study. The F3b females were examined grossly and discarded after weaning of the F4c litters and the F3b males were sacrificed, examined grossly, with weights recorded for testes, adrenals and seminal vesicles. These organs plus brain and thyroid were preserved for microscopic examination. Examination of the F3b females revealed resorption sites in some of the rats which had failed to produce litters or had produced very small litters: one control female, and two low dose females had such resorbing foetuses. In addition, one control female and one low dose female died immediately after giving birth. One high dose female died before giving birth and was noted to have term foetuses. Ovarian cysts were noted in one control and one high dose female, but these rats had normal litters. Organ weights and ratios of the F3b males varied considerably as did body weights. Ratios for seminal vesicles were comparable in all of the groups but organ weights and ratios of testes and adrenals were less in the test diet groups than in the controls. Those rats which were ineffective in fathering litters had reduced testes weights which lowered the average for the groups. Reduced adrenal weights were not related to sterility, however. In general, the same male rats which had not fathered F4a litters were ineffective in fathering F4b and F4c litters. Histopathological findings showed very good correlation with inability to father litters; every male which had failed to father litters in both the F4a and F4c generations had testicular pathology including degeneration of seminiferous epithelium with or without fibrosis, or lobular degeneration. No testicular pathology wan seen in fertile males and no other organs showed pathology in any of the males.

- Although the numbers of F4c litters produced were less in the test diet groups than in the control group, size and weights of litters were comparable for all groups. Mortality was somewhat higher in the test diet groups than in the control. The F4c weanlings from the control, low and high groups, however, were maintained on the diets and mated when 120 days old.

- Both F5a and F5b litters were normal in number, size, weight and percent survival in all groups. Parent rats warts comparable at sacrifice and no abnormalities were noted at autopsy in either parents or pups. No tissues were taken for histology and the study was terminated at this point.

Conclusions:

Under the conditions of this study, the interpretation of the reproductive studies was rendered difficult because of several changes in the mortality pattern, however 50 ppm concentration of the test material in the diet might be considered to be the test material effect level in that possibly resistance to infection was lowered in both mothers and weanlings. There were no effects from the test material seen in the adult weights, weanling weights, organ/ body weight ratios in weanlings or pathology.

Executive summary:

The reproductive toxicity of the test material was investigated in a three generation reproductive study, performed on male and female Long Evans rats using a non-standard method.

Animals were treated with the test material in their diet at 20, 50 and 100 ppm, they were mated on the 79th day of treatment when they were at least 100 days old. Each female was exposed to two different males during a two week mating period, all pups from the first litter born were discarded at weaning and the parent animals were mated again ten days later. The protocol was followed in this way for three generations. Litters greater than 10 in number were reduced to ten on the fifth day by selecting runts, where possible, to discard. On day 21 the weanlings were counted and weighed and either sacrificed or saved for continuation on the diets.

There were no differences among the groups in the adult weights, weanling weights, organ/ body weight ratios in weanlings or pathology.

Interpretation of the reproductive studies was rendered difficult because of several changes in the mortality pattern. This first occurred among pregnant and nursing females and 3 to 4 weeks old weanlings in the groups receiving the two highest doses of test material. However, unexpected mortality in the later meetings occurred as well in the control group. If an extraneous infection was in fact present in the colony, it is possible that the rats receiving the higher dose levels of test material would have been the first to succumb, due to the extra stress. On this basis, 50 ppm of the test material might be considered to be the test material effect level in that possibly resistance to infection was lowered in both mothers and weanlings. The low production of litters in several of the groups in later generations was attributed to sterility in some of the males which also may have resulted from the same infection.

As a result of the changing mortality pattern, two supplementary/ follow on studies were performed. Continuation of the reproduction study further confirmed the difficulty encountered in the F2 and F3 generations was attributable to male rats. Litter production by the F3b rats was below normal in all test groups at the first mating, but the majority of females which failed to produce F4a litters were successful in producing F4b litters when mated with normal males. Most males which had been ineffective in producing F4a litters were ineffective also when mated with normal females. Microscopic examination of the testes of all F3b males revealed degenerative pathology in those males known to be non-fertile. The etiology of this degenerative change is not clear but it does not appear to be treatment related.

The addition of Brewers yeast to the diets had no significant effect on fecundity of the F3b rats or survival of their litters: litter production (F4c’s) was not improved over that seen prior to receiving the fortified diets and mortality was actually higher among F4c’s than among F4a’s. It should be noted that the F3b groups had been on the pelletised non-fortified diet until just prior to breeding for F4c litters and the degenerative pathology was apparently already present. Only the F4c groups were entirely on a fortified powdered diet. Among the F4c rats used for subsequent breeding, however, there was no evidence of reduced fertility or increased mortality in any of the groups, and the F5a and F5b litters were all within normal limits. In conclusion the continued study of the infertility pattern noted in the earlier portion of the reproduction study revealed a reversal in the trend, with normal fecundity achieved by the fifth generation. There was correlation between testicular pathology and lack of fertility in F3b males.

Under the conditions of this study, the interpretation of the reproductive studies was rendered difficult because of several changes in the mortality pattern, however 50 ppm concentration of the test material in the diet might be considered to be the test material effect level in that possibly resistance to infection was lowered in both mothers and weanlings. There were no effects from the test material seen in the adult weights, weanling weights, organ/ body weight ratios in weanlings or pathology, however there was correlation between testicular pathology and lack of fertility in F3b males.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

1.5 mg/kg bw/day

Study duration:

chronic

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

2 Generation Reproductive Toxicity Study (mixture DOTI: MOTI. ca. 80:20) (CAS No 26401-97-8 and CAS 26401-86-5)

The reproductive toxicity of the test material was investigated in a two-generation reproduction toxicity test conducted in accordance with the standardised guideline OECD 416 under GLP conditions using rats. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

During the study, the test material was administered to the animals through their diet which was available to them ad. libitum at 20, 60 or 200 ppm. The concentrations of the doses were analytically verified. 25 animals per sex per dose were treated at each concentration.

The F0-generation animals were exposed to the appropriate test diets seven days/week for a 70-day (10-week) premating treatment period. The male and female rats continued to be treated daily during the 21-day mating period and the females continued to be treated during the ensuing gestation and lactation periods. The F1-generation animals received the appropriate diet (dose-level of the parent animals of the F0-generation) seven days/week for a 98-day (14-week) premating period. The male and female rats continued to receive the appropriate test diets during the 21-day mating period and females for the gestation and lactation periods similar to the animals of the F0-generation.

The no-observed-effect-level (NOEL) for the males and females of the F0-generation was 20 ppm (approx. 1.5 mg/kg bw/day). Starting at 60 ppm (approx. 4.4 mg/kg bw/day) a slightly reduced relative thymus weight for the males was observed. At 200 ppm (approx. 14.7 mg/kg bw/day) the incidence of thymic involution was increased. The changes of the thymus represent an expected well-known effect of compounds in the test material.

The no-observed-effect level (NOEL) for the F1-generation until weaning was 20 ppm (approx. 1.6 mg/kg bw/day). At 60 ppm (approx. 4.7 mg/kg bw/day) the relative thymus weight was decreased in male (slightly) and female pups (significantly). At 200 ppm (approx. 15.9 mg/kg bw/day) the relative thymus weight was significantly reduced in both sexes. The body weight of the pups at birth was slightly reduced and the body weight gain was distinctly inhibited. For the F1-generation post lactation, the no effect dose-level was also 20 ppm (approx. 1.6 mg/kg bw/day). Starting at 60 ppm (approx. 4.7 mg/kg bw/day) a slightly reduced relative thymus weight was observed for the males. In addition, a suspected increase in still births was observed. 200 ppm (approx. 15.9 mg/kg bw/day) caused moderate toxicity such as reduced body weight and reduced food consumption and influenced reproduction parameters (increased number of stillbirths, decreased viability index, decrease in pup weights). The relative thymus weight of males and females was significantly below the controls and the incidence of thymic involution was increased in the male animals.

In all treated groups no effects on organ weights nor histopathological changes were observed in the F2-generation. No teratogenic effect was observed.

No influence on reproduction parameters were observed at 20 and 60 ppm in F0 and F1 generation. There was a slight increase in stillbirths at 60 ppm (F1) which was suspected to be treatment-related.

Under the conditions of the study the NOEL of the test material for reproduction in the F0 and F1 generations was 20 ppm in the diet (equivalent to approximately 1.5 - 1.6 mg/kg bw/day).

3-generation reproductive study

The reproductive toxicity of the test material was investigated in a three generation reproductive study, performed on male and female Long Evans rats using a non-standard method.

Animals were treated with the test material in their diet at 20, 50 and 100 ppm, they were mated on the 79th day of treatment when they were at least 100 days old. Each female was exposed to two different males during a two week mating period, all pups from the first litter born were discarded at weaning and the parent animals were mated again ten days later. The protocol was followed in this way for three generations. Litters greater than 10 in number were reduced to ten on the fifth day by selecting runts, where possible, to discard. On day 21 the weanlings were counted and weighed and either sacrificed or saved for continuation on the diets.

There were no differences among the groups in the adult weights, weanling weights, organ/ body weight ratios in weanlings or pathology.

Interpretation of the reproductive studies was rendered difficult because of several changes in the mortality pattern. This first occurred among pregnant and nursing females and 3 to 4 weeks old weanlings in the groups receiving the two highest doses of test material. However, unexpected mortality in the later meetings occurred as well in the control group. If an extraneous infection was in fact present in the colony, it is possible that the rats receiving the higher dose levels of test material would have been the first to succumb, due to the extra stress. On this basis, 50 ppm of the test material might be considered to be the test material effect level in that possibly resistance to infection was lowered in both mothers and weanlings. The low production of litters in several of the groups in later generations was attributed to sterility in some of the males which also may have resulted from the same infection.

As a result of the changing mortality pattern, two supplementary/ follow on studies were performed. Continuation of the reproduction study further confirmed the difficulty encountered in the F2 and F3 generations was attributable to male rats. Litter production by the F3b rats was below normal in all test groups at the first mating, but the majority of females which failed to produce F4a litters were successful in producing F4b litters when mated with normal males. Most males which had been ineffective in producing F4a litters were ineffective also when mated with normal females. Microscopic examination of the testes of all F3b males revealed degenerative pathology in those males known to be non-fertile. The etiology of this degenerative change is not clear but it does not appear to be treatment related.

The addition of Brewers yeast to the diets had no significant effect on fecundity of the F3b rats or survival of their litters: litter production (F4c’s) was not improved over that seen prior to receiving the fortified diets and mortality was actually higher among F4c’s than among F4a’s. It should be noted that the F3b groups had been on the pelletised non-fortified diet until just prior to breeding for F4c litters and the degenerative pathology was apparently already present. Only the F4c groups were entirely on a fortified powdered diet. Among the F4c rats used for subsequent breeding, however, there was no evidence of reduced fertility or increased mortality in any of the groups, and the F5a and F5b litters were all within normal limits. In conclusion the continued study of the infertility pattern noted in the earlier portion of the reproduction study revealed a reversal in the trend, with normal fecundity achieved by the fifth generation. There was correlation between testicular pathology and lack of fertility in F3b males.

Under the conditions of this study, the interpretation of the reproductive studies was rendered difficult because of several changes in the mortality pattern, however 50 ppm concentration of the test material in the diet might be considered to be the test material effect level in that possibly resistance to infection was lowered in both mothers and weanlings. There were no effects from the test material seen in the adult weights, weanling weights, organ/ body weight ratios in weanlings or pathology, however there was correlation between testicular pathology and lack of fertility in F3b males.

Effects on developmental toxicity

Description of key information

Deficiencies in the results from the the registered substance (DOTI) data mean that the conclusions are to be taken from the read across substance (DOTE, CAS 15571-58-1).

Read-across to (DOTE) (CAS No 15571 -58 -1).

Developmental Toxicity Study in Mice and Rabbits, (2014)

- Mouse: The NOAEL for maternal toxicity was determined to be 15 mg/kg based on a biologically relevant depression in thymus size at the 30 mg/kg dose. However, no treatment-related effects were noted in any of the developmental toxicity parameters investigated; therefore the NOAEL for developmental toxicity was concluded to be 60 mg/kg, the highest dose tested.

- Rabbit: The NOAEL for maternal toxicity was determined to be 20 mg/kg based on a biologically relevant depression in thymus weight at the 80 mg/kg dose. However, no treatment-related effects were noted in any of the developmental toxicity parameters investigated; therefore the NOAEL for developmental toxicity was concluded to be 80 mg/kg, the highest dose tested.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 August 2014 to 11 November 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

other: read-across target

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

mouse

Strain:

Swiss

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: In-house bred animals
- Age at study initiation: 10 - 12 weeks
- Weight at study initiation (GD 0): 24.01 - 33.20 g
- Housing: during the pre-mating period a maximum of three animals were housed in a standard polypropylene cage (290 x 220 x 140 mm) with stainless steel mesh top grill. Clean sterilised paddy husk was provided as bedding material. During the mating period two mice (one male and one female) were housed together until confirmation of mating (GD 0) at which point the females were housed individually in polypropylene cages and males were housed with their former cage mates.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6 - 23.1 °C
- Humidity (%): 50 - 59 % (relative)
- Air changes (per hr): 12 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

Route of administration:

oral: gavage

Vehicle:

peanut oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test material was administered by oral gavage in peanut oil at a dose volume of 5 mL/kg bw. Control animals received vehicle only at a dose volume of 5 mL/kg bw.
Fresh dosing solutions were prepared daily before administration.

VEHICLE
- Justification for use and choice of vehicle: Peanut oil was selected since it has been utilised in previous toxicology studies with related test materials and is a routinely used vehicle.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Formulation analysis was conducted to enable verification of dose concentration by analysing total tin content. Analyses were conducted during week 1 and week 2 of the dosing period. The dose samples were collected in duplicate (2 x 5 mL) for each dose formulation including vehicle control. One set of samples were analysed while the other set was stored and analysed later, if required.

Details on mating procedure:

- Impregnation procedure: Cohoused
- If cohoused:
- M/F ratio per cage: 1 male / 1 female
- Length of cohabitation: Until copulation was confirmed or for 2 weeks (maximum)
- Proof of pregnancy: Vaginal plug and/or vaginal smear, referred to as day 0 of pregnancy

Females not mated within 14 days of pairing with the first male were placed with a second proven male if presence of sperm in the vaginal smear and/or vaginal plug was not confirmed.

Duration of treatment / exposure:

Females received test material daily from gestation day 5 to gestation day 17 (inclusive).

Frequency of treatment:

Daily throughout treatment period.

Duration of test:

Dams were sacrificed on gestation day 18.

No. of animals per sex per dose:

25 pregnant females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses were selected upon consideration of the following available data.
In a published report of an oral gavage developmental toxicity study in mice [Faqi, 2001] with a closely related dioctyltin material [dioctyltin isooctyl thioglycolate; DOT[IOTG]; DOTI], the test material utilised had approximately 80 % dioctyl and 20 % monooctyl components. Doses were 20, 30, and 45 mg/kg body weight [Phase I], and 67 and 100 mg/kg body weight [Phase II]. Dosing was done from gestation day 6 [GD6; day of vaginal coital plug = GD1] to gestation day 17 [GD17]. Caesarean sections were done on GD18.
Adjusted maternal weight gain [GD18 weight minus GD1 weight minus gravid uterine weight] at the 100 mg/kg dose was 56 % of the control. High variability in the maternal weight data, as evidenced by the high SD of the mean, obscured the statistical significance of this biologically relevant maternally toxic outcome. One maternal death occurred at this dose further supporting the fact that this is a maternally toxic dose. The maternal weight effect was accompanied by a statistically significant decrease in maternal thymus weight. Maternal liver weight, on both an absolute and relative basis, was also statistically significantly decreased at this dose.
Relative to the control mice, maternal thymus weight and liver weight trended downward at 67 mg/kg, suggesting that this dose is an upper bound in order to preclude an unacceptable level of maternal toxicity and generate a valid assessment of developmental toxicity in a new study.
The number of dams maintaining pregnancy in the treated groups up to the 45 mg/kg dose was not different from controls. The number of implantation sites, number of viable foetuses, and foetal weight were unaffected by treatment. There was a suggestion of an increase in the number of resorptions at 45 mg/kg, but this was a not statistically significant and the number of viable foetuses was comparable to controls at this dose.
The number of dams maintaining pregnancy in the control group for Phase II of the study was decreased relative to all other groups in the study. The number of dams maintaining pregnancy in the 67 and 100 mg/kg groups was not statistically different than the Phase II control. As noted above, the 67 mg/kg dose appears to be the upper bound dose for a developmental toxicity study because foetal weight at the 67 mg/kg dose is significantly less than controls, the number of resorptions is increased, and the number of resorptions per implantation is statistically significantly increased.
There is evidence of foetal toxicity, expressed as delayed ossification, at 45 mg/kg and higher which further supports 67 mg/kg as an upper bound for any further investigation of developmental toxicity.
In the study reported by Henninghausen, (1979) a single intramuscular dose of dioctyltin chloride [DOTC] at 60 mg/kg reduced thymus weight to 48 % of controls and thymocyte count to 64 % of controls. A single oral gavage dose of 300 mg/kg resulted in thymus weight at 65 % of controls and thymocyte count at 70 % of controls. The repeated-dose test paradigm for developmental toxicity studies makes it highly unlikely that pregnancy in mice could be sustained.
Therefore, the high dose chosen for this study is 60 mg/kg, to reflect a dioctyltin dose with minimal maternal and foetal toxicity as the upper bound. It is anticipated that this dose will meet the developmental toxicity test guideline criteria of producing some maternal toxicity without compromising the survival of the pregnant dam, the integrity of pregnancies to Day 18, or the survival of the developing foetuses.
The low dose chosen (15 g/kg) was selected to reflect the NOEL in the published study.

- Rationale for animal assignment: Each day the body weight of mated mice was recorded. The mated females were distributed to all groups based on their body weights so as to maintain comparable mean body weights across all groups.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: animals were observed twice daily for mortality and morbidity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: animals were observed once daily for clinical signs of toxicity.

BODY WEIGHT: Yes
- Time schedule for examinations: Dams were weighed on gestation days 0, 3, 5 and daily thereafter up to, and including, gestation day 18.

FOOD CONSUMPTION: Yes
- Time schedule: individual feed intake of mated females was recorded on gestation days 0 - 3, 3 - 5, 5 - 7, 7 - 9, 9 - 11, 11 - 14, 14 - 17, and 17 - 18.

WATER CONSUMPTION: No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 18
All the animals were sacrificed on gestation day 18 by carbon dioxide exposure and subjected to detailed gross pathology. At necropsy the thymus was excised, weighed and placed in 10 % formalin for possible future histopathological examination.

Ovaries and uterine content:

The ovaries and uterine content were examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Number of live and dead foetuses: Yes

Fetal examinations:

- External examinations: Yes
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
- Head examinations: Yes

Other examinations included:
- Sex, number and weight of live foetuses
- Crown rump length (measured after sacrifice)

Statistics:

Raw data was subjected to computer statistical processing. One-way ANOVA with Dunnett's post test was performed for data (body weight, food consumption, number of corpora lutea, uterus weight, number of implantations, number of live foetuses, body weight of live foetuses), using SPSS software. All analyses and comparisons were evaluated at the 95 % level of confidence (P<0.05).

Indices:

Corrected body weight (g) = (Gestation day 18 body weight – Gestation day 5 body weight) – Gravid uterus weight
Pre-implantation loss (%) = ((Number of corpora lutea – Number of implants)/Number of corpora lutea) x 100
Post-implantation loss (%) = ((Number of implants – Number of viable foetuses)/Number of Implants) x 100
Male/Female sex ratio = Number of live male foetuses/Number of live female foetuses
Male/female foetuses (%) = (Number of live male foetuses/Total number of foetuses) x 100 or (Number of live female foetuses/Total number of foetuses) x 100
Foetal incidence (%) = (Number of foetuses with a particular observation/Total number of foetuses in a group) x 100

Clinical signs:

no effects observed

Description (incidence and severity):

The animals did not reveal any clinical signs of toxicity and mortality at any of the tested doses throughout the experiment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There were no statistically significant differences in maternal body weights across the dose groups on any single gestation day. However, there was a clear dose-related pattern of reduced body weights beginning after GD6, the first day of dosing, and continuing for the duration of the study. The high dose was the most severely affected, though a dose-related decrement relative to the control body weights can be seen across all doses particularly from GD16 to GD18. At 30 mg/kg the maternal weight effect was marginal, but maternal body weight gain in the 60 mg/kg high dose group was 11.5 % [uncorrected] and 26.6 % [corrected] less than the vehicle control, a clear adverse effect. The corrected body weight gain in the low (15 mg/kg) and mid (30 mg/kg) dose groups were -16.7 % and - 17.7 % when compared to the controls.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No treatment related differences in average feed consumption were observed at any dose.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

There was a treatment-related macroscopic finding of reduced maternal thymus weight.
The mean maternal thymus weight was statistically significantly reduced in the 30 mg/kg [mid] and 60 mg/kg [high] dose groups. The mean maternal thymus weight in the low dose mice was reduced relative to controls, but was not statistically significant. These observations are indicative of a treatment-related specific target organ toxicity resulting from exposure to the test material. No other gross pathological findings were noted in any dose group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

There was a treatment-related macroscopic finding of reduced maternal thymus weight.
The mean maternal thymus weight was statistically significantly reduced in the 30 mg/kg [mid] and 60 mg/kg [high] dose groups. The mean maternal thymus weight in the low dose mice was reduced relative to controls, but was not statistically significant. These observations are indicative of a treatment-related specific target organ toxicity resulting from exposure to the test material. No other gross pathological findings were noted in any dose group.

Dead fetuses:

no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

A total number of 21 (84 %), 21 (84 %), 20 (80 %) and 20 (80 %) mated females were confirmed pregnant at the time of caesarean section for groups G1, G2, G3 and G4, respectively.

Other effects:

no effects observed

Description (incidence and severity):

There were no statistically significant differences in these gravid uterus weights, number of implantation sites, pre- and post-implantation loss or early or late resorptions across dose groups when compared to the vehicle control.

Dose descriptor:

NOAEL

Effect level:

15 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

organ weights and organ / body weight ratios

other: Statistically significant decrease in thymus size at 30 mg/kg

Dose descriptor:

LOAEL

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

organ weights and organ / body weight ratios

other: Statistically significant depression in thymus size

Dose descriptor:

NOAEL

Effect level:

60 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The mean foetal weights [combined sexes] were 1.35, 1.37, 1.30 and 1.31 grams for groups G1, G2, G3 and G4, respectively. Mean foetal weights were not statistically significantly different across the dose groups when compared to controls.

External malformations:

no effects observed

Description (incidence and severity):

No external abnormalities were noted during gross examinations of foetuses at any dose.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The noted anomalies (poorly ossified frontal, parietal and inter-parietal bones; ossification site at first lumbar vertebrae; supplementary ribs) are common findings for foetuses of this species and strain. These morphologic observations did not occur in a dose-dependent pattern, nor was the severity of the anomaly increased with dose. The findings were therefore considered to be incidental, and not indicative of a teratogenic effect.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related abnormalities were observed during visceral examinations of foetuses at any dose. The noted findings [pale coloured kidneys and dilated renal pelvis] are common findings for foetuses of this species and strain. The observations were not dose dependent, nor was the severity of the anomaly increased with dose. This result supports the conclusion that the findings are incidental and that the test material did not produce an adverse effect during foetal development of the soft tissues.

Description (incidence and severity):

There were no statistically significant differences in the sex ratio, mean litter size or the number of live foetuses per dam across dose groups when compared to the vehicle control.
- Crown-rump length
The average crown-rump lengths were 23.2, 24.0, 23.3 and 22.9 mm for groups, G1 through G4, respectively. There were no statistically significant differences in length across all dose groups when compared to the control.

Remarks on result:

not determinable due to adverse toxic effects at highest dose / concentration tested

Abnormalities:

not specified

Developmental effects observed:

not specified

Dose Formulation Analysis

The results for chemical homogeneity and dose concentration verification are within the acceptance limits of ± 10 % of the nominal concentrations.

Table 1: Maternal Body Weights (g)

Dose Group (mg/kg)	Gestation Day
	0	3	5	6	7	8	9	10	11	12	13	14	15	16	17	18
G1: 0	27.41	28.25	29.03	29.49	30.06	30.54	31.41	32.68	34.03	35.59	37.52	39.34	41.31	43.81	46.23	49.10
G2: 15	27.08	27.71	28.33	28.90	29.59	30.21	30.74	31.73	33.20	34.80	36.73	38.81	41.12	43.96	46.02	48.21
G3: 30	27.23	28.11	28.67	29.20	29.59	30.42	31.04	32.24	33.72	35.13	36.80	38.76	40.78	43.35	45.71	48.17
G4: 60	26.93	27.69	28.58	29.12	29.52	30.11	30.74	31.74	33.06	34.61	36.15	37.81	40.15	42.63	44.30	46.35

 

Table 2: Maternal Body Weight Gain

Dose Group (mg/kg)	GD 5-18 (g)	GD 5-18 (% of control)	GD 5-18 (% gain compared to control)
G1: 0	20.07	100.0	0.0
G2:15	19.18	95.6	-4.4
G3: 30	19.5	97.2	-2.8
G4: 60	17.77	88.5	-11.5

 

Table 3: Litter Data

Dose Group	No. of Corpora Lutea (mean)	Implantation Sites (mean)	Live Foetuses (mean)	No. of Early Resorptions (mean)	No. of Late Resorptions (mean)	Mean Pre-implantation Loss (%)	Mean Post-implantation Loss (%)
G1: 0	10.1	10.1	10.1	0.0	0.0	0.0	0.0
G2: 15	10.7	10.7	10.1	0.1	0.0	0.0	0.9
G3: 30	10.7	10.6	10.5	0.1	0.1	0.5	1.5
G4: 60	10.0	10.0	9.7	0.1	0.2	0.0	2.6

 

Group & Dose (mg/kg)	Mean Thymus weight (g)
G1 (0 mg/kg)	0.0400
G2 (15 mg/kg)	0.385
G3 (30 mng/kg)	0.0306*
G4 (60 mg/kg)	0.0260*

* p < 0.05

 

Table 4: Summary of Foetal Examinations

Parameters	Group (Dose mg/kg)	G1 (0 mg/kg)	G2 (15 mg/kg)	G3 (30 mg/kg)	G4 (60 mg/kg)
External Examinations
Malformations	No. of foetuses	0	0	0	0
	%	0.0	0.0	0.0	0.0
Variations	No. of foetuses	0	0	0	0
	%	0.0	0.0	0.0	0.0
Visceral Examinations
Malformations	No. of foetuses	0	0	0	0
	%	0.0	0.0	0.0	0.0
Variations	No. of foetuses	4	5	3	6
	%	4.0	4.8	3.0	6.5
Skeletal
Malformations	No. of foetuses	0	0	0	2
	%	0.0	0.0	0.0	2.0
Variations	No. of foetuses	6	3	2	9
	%	5.4	2.6	2.0	8.8

 

Conclusions:

Under the conditions of the study the NOAEL for maternal toxicity was determined to be 15 mg/kg based on a biologically relevant depression in thymus size at the 30 mg/kg dose. However, no treatment-related effects were noted in any of the developmental toxicity parameters investigated; therefore the NOAEL for developmental toxicity was concluded to be 60 mg/kg, the highest dose tested.

Executive summary:

The purpose of this study was to assess the effects of prenatal exposure of pregnant female Swiss mice and their developing foetuses to the test material when administered by oral gavage in a peanut oil vehicle at 5 mL/kg to the mated females from gestation day [GD] 5 to 17. In life examinations included checks for mortality and clinical signs of toxicity, body weight measurements and an evaluation of food consumption. All the animals were sacrificed on gestation day 18 by carbon dioxide exposure and subjected to detailed gross pathology; the gross pathology included a determination of thymus size. The gravid uterus was collected by hysterectomy and foetuses were removed by caesarean section. Foetuses were subjected to external, soft tissue and skeletal examination.

No deaths were observed during the experimental period and there were no clinical signs recorded which were indicative of overt toxicity. There were no statistically significant differences in maternal body weights across the dose groups on any single gestation day. However, there was a clear dose-related pattern of reduced body weights beginning after GD6, the first day of dosing, and continuing for the duration of the study. The high dose was the most severely affected and a dose-related decrement relative to the control body weights can be seen across all doses particularly from GD16 to GD18. At 30 mg/kg the maternal weight effect was marginal, but maternal body weight gain in the 60 mg/kg high dose group was 11.5 [uncorrected] and 26.6 % [corrected] less than the vehicle control, a clear adverse effect. There was a treatment-related macroscopic finding of reduced maternal thymus weight.

The mean maternal thymus weight was statistically significantly reduced in the 30 mg/kg [mid] and 60 mg/kg [high] dose groups. The mean maternal thymus weight in the low dose mice was reduced relative to controls, but was not a statistically significant difference. No other gross pathological findings were noted in any dose group.

No treatment related effects were noted in mean gravid uterus weight, no. of corpora lutea, no. of implantations in all the groups, no. of early or late resorptions and percentage of post implantation loss.

No external abnormalities were noted during gross examinations of foetuses at any dose and no treatment-related abnormalities were observed during visceral examinations of foetuses at any dose.

Therefore, under the conditions of the study the NOAEL for maternal toxicity was determined to be 15 mg/kg based on a biologically relevant depression in thymus size at the 30 mg/kg dose. However, no treatment-related effects were noted in any of the developmental toxicity parameters investigated; therefore the NOAEL for developmental toxicity was concluded to be 60 mg/kg, the highest dose tested.

Reference 2

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted on read-across material

Justification for type of information:

Read-across to structurally similar substance Dioctyltin bis (2-ethylhexylmercaptoacetate) (DOTE) (CAS No. 15571-58-1), see attached justification.

Reason / purpose for cross-reference:

read-across source

Dose descriptor:

NOAEL

Effect level:

15 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

organ weights and organ / body weight ratios

other: Statistically significant decrease in thymus size at 30 mg/kg

Dose descriptor:

LOAEL

Effect level:

30 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

organ weights and organ / body weight ratios

other: Statistically significant depression in thymus size

Dose descriptor:

NOAEL

Effect level:

60 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Remarks on result:

not determinable due to adverse toxic effects at highest dose / concentration tested

Developmental effects observed:

not specified

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 July 2014 to 12 November 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

other: read-across target

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

-TEST ANIMALS
- Age at study initiation: 5 - 6 months
- Weight at study initiation (GD 0): 2.50324 - 3.74756 kg
- Housing: during the pre-mating period animals were housed individually in stainless steel wire mesh cages (24 x 18 x 18 inches). During the mating period two rabbits (one male and one female) were housed together until visual confirmation of mating (GD 0). The mated rabbits were housed in their original cage.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6 - 22.6 °C
- Humidity (%): 50 - 63 % (relative)
- Air changes (per hr): 12 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

Route of administration:

oral: gavage

Vehicle:

peanut oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test material was administered by oral gavage in peanut oil at a dose volume of 2 mL/kg. Control animals received vehicle only at a dose volume of 2 mL/kg.
Fresh dosing solutions were prepared daily before administration.

VEHICLE
- Justification for use and choice of vehicle: peanut oil was selected since it has been utilised in previous toxicology studies with related test materials and is a routinely used vehicle.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Formulation analysis was conducted to enable verification of dose concentration by analysing total tin content. Analyses were conducted during week 1 and week 6 of the dosing period. The dose samples were collected in duplicate (2 x 5 mL) for each dose formulation including vehicle control. One set of samples were analysed while the other set was stored and analysed later, if required. The acceptance criteria for dose concentration test was ± 10 % recovery to the nominal concentration.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1 male/ 1 female
- Length of cohabitation: animals were cohoused until evidence of copulation was observed visually
- Proof of pregnancy: Visual observation of copulation; referred to as day 0 of pregnancy

Following the acclimatisation period of 4 - 6 female rabbits were paired per day with males.
In case of unsuccessful mating, females were returned to their original cages and given an opportunity to mate at a later time.

Duration of treatment / exposure:

Daily from gestation day 6 to gestation day 28 (inclusive).

Frequency of treatment:

Daily throughout treatment period.

Duration of test:

Does were sacrificed on gestation day 29.

No. of animals per sex per dose:

24 presumed-pregnant females per dose

Control animals:

yes

Details on study design:

- Dose selection rationale: Doses were selected upon consideration of available data on a closely related dioctyltin compound [dioctyltin iso-octyl thioglycolate; DOT[IOTG]; DOTI]. Two previous oral gavage developmental toxicity studies in the rabbit are available. Both used peanut oil as a vehicle and the test material utilised for both had approximately 80 % dioctyl and 20 % monooctyl components.
The first study with DOTI is a range-finding study using doses of 3, 10 and 30 mg/kg administered from gestation day 6 [GD6] to gestation day 18 [GD18]. Caesarean sections were done on GD28. In addition, within this study a dose of 100 mg/kg was added because there were no apparent maternal effects at doses up to and including 30 mg/kg. There were no observed developmental effects among the foetuses from treated does at doses up to and including 30 mg/kg. At the 100 mg/kg there was a decrease in maternal body weight and a possible increase in the incidence of dead foetuses.
The second study with DOTI is a definitive developmental toxicity study which followed the same methodology as the range-finding study. Doses were 1, 10, and 100 mg/kg. In the second study clear maternal toxicity was observed in the 100 mg/kg dose and developmental effects consistent with delayed development of the foetuses [low weight, delayed ossification]. The noted anomalies were classified as minor anomalies and the low incidence of major anomalies could not be definitively attributed to treatment with the test material.
In the definitive study with DOTI dosing was limited to GD6-GD18; there were 3 maternal deaths and 4 abortions at the 100 mg/kg dose. The maternal deaths could not definitively be ascribed to treatment, but the abortions were determined to be the result of treatment. These results are relevant to the selection of the high dose in the current study; the extended dosing regimen of GD6 - GD28 and the higher proportion of the dioctyl component may increase maternal toxicity beyond what was observed with DOTI and is considered to possibly exceed the upper bound dose for a study of this type. Therefore the maximum dose tested is 80 mg/kg bw/day.

- Rationale for animal assignment: Each day the body weight of mated rabbits was recorded. The mated females were distributed to all groups based on their body weights so as to maintain comparable mean body weights across all groups.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: animals were observed twice daily for mortality and morbidity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: animals were observed once daily for clinical signs of toxicity.

BODY WEIGHT: Yes
- Time schedule for examinations: Does were weighed on gestation days 0, 3, 6 and daily thereafter until gestation day 29.

FOOD CONSUMPTION: Yes
- Time schedule: Individual feed intake of mated females was recorded on gestation days 0 - 3, 3 - 6 and daily from day 6 to day 29.

WATER CONSUMPTION: No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
All the animals were sacrificed on gestation day 29 by intravenous injection of sodium thiopentone and subjected to detailed gross pathology. At necropsy the thymus was excised, weighed and placed in 10 % formalin for possible future histopathological examination. Furthermore, all gross lesions were excised and placed in 10 % formalin for possible future histopathological examination.

Ovaries and uterine content:

The ovaries and uterine content were examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Number of live and dead foetuses: Yes

Fetal examinations:

- External examinations: Yes
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
- Head examinations: No data

Other examinations included:
- Sex, number and weight of live foetuses
- Crown rump length (measured after sacrifice)

Statistics:

Raw data was subjected to computer statistical processing. One-way ANOVA with Dunnett's post test was performed for body weight, food consumption, number of corpora lutea, uterus weight, number of implantations, number of live foetuses and body weight of live foetuses. Kruskal-Wallis test followed by Dunnett's post test was used for the percentage of visceral or skeletal malformations (or variations) for each litter, percentage of each individual malformation (or variation) for each litter using SPSS software version 2. All analyses and comparisons were evaluated at the 95 % level of confidence (P<0.05).

Indices:

Corrected body weight (g) = (Gestation day 29 body weight - Gestation day 6 body weight) - Gravid uterus weight
Pre-implantation loss (%) = ((Number of corpora lutea - Number of implants)/Number of corpora lutea) x 100
Post-implantation loss (%) = ((Number of implants - Number of viable foetuses)/Number of implants) x 100
Male/female sex ratio = Number of live male foetuses/Number of live female foetuses
Male/female foetuses (%) = (Number of live male foetuses/Total number of foetuses) x 100 or (Number of live female foetuses/Total number of foetuses) x 100
Foetal incidence (%) = (Number of foetuses with a particular observation/Total number of foetuses in a group) x 100

Mortality:

no mortality observed

Description (incidence):

No deaths or abortions were observed during the experimental period and there were no clinical signs recorded which were indicative of overt toxicity.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no statistically significant differences in maternal body weights across the dose groups on any single gestation day, nor was there a dose-related pattern of reduced body weights beginning after GD6, the first day of dosing.There were no statistically significant differences in uncorrected maternal body weigh gain across the dose groups for any single gestational period. Treated does had higher weights than controls on GD29, this was considered to be a random occurrence and not related to test material administration.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No treatment related differences in average feed consumption were observed at any dose.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

There was a treatment-related macroscopic finding of reduced maternal thymus weight. The mean maternal thymus weights were -5.1, -9.6, and -12.8 % in the 4 mg/kg [low], 20 mg/kg [mid], and 80 mg/kg [high] dose groups, respectively when compared to controls. While these decreases were not statistically significant, the results were dose-dependent relative to controls and are consistent with data from other species [rat and mouse] which demonstrate the thymus is a target organ for octyltin compounds. These observations are indicative of a treatment-related specific target organ toxicity resulting from exposure to the test material. No other gross pathological findings were noted in any dose group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

There was a treatment-related macroscopic finding of reduced maternal thymus weight. The mean maternal thymus weights were -5.1, -9.6, and -12.8 % in the 4 mg/kg [low], 20 mg/kg [mid], and 80 mg/kg [high] dose groups, respectively when compared to controls. While these decreases were not statistically significant, the results were dose-dependent relative to controls and are consistent with data from other species [rat and mouse] which demonstrate the thymus is a target organ for octyltin compounds. These observations are indicative of a treatment-related specific target organ toxicity resulting from exposure to the test material. No other gross pathological findings were noted in any dose group.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The mean pre-implantation losses were 0.9, 0.8, 2.3, and 4.9 % for groups G1, G2, G3 and G4, respectively. Mean post-implantation losses for these treatment groups were 3.1, 3.5, 6.4 and 5.7, respectively. These losses are within the historical control range and there is no clear evidence of a dose-response across dose groups.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

A total number of 19, 21, 19 and 20 mated females were confirmed pregnant in groups G1, G2, G3 and G4, respectively (0, 4, 20 and 80 mg/kg, respectively). This is a pregnancy rate of 79, 88, 79 and 83 % at the time of caesarean section for groups G1, G2, G3 and G4, respectively.

Other effects:

no effects observed

Description (incidence and severity):

No treatment related effects were noted in the number of corpora lutea, number of implantation sites, number of early or late resorptions, or percentage of post implantation loss across all the groups.
The mean gravid uterus weights were 230.1, 309.5, 253.7 and 196.9 g for groups G1 through G4, respectively. A statistically significant increase in the mean gravid uterus weight was noted at the low dose when compared to the vehicle control. This difference was attributed to the increase in the number of foetuses compared to the vehicle control group and other dose groups.

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

organ weights and organ / body weight ratios

other: maternal toxicity

Dose descriptor:

LOAEL

Effect level:

80 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

organ weights and organ / body weight ratios

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

80 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The mean foetal weights [combined sexes] were 36.6, 37.3, 35.5, and 32.3 g for groups G1, G2, G3 and G4, respectively. Mean foetal weights were not statistically significantly different across the dose groups when compared to controls. At the high dose the mean foetal body weight was -11.9 % relative to controls which suggests a biologically-relevant, but marginal effect on foetal maturation. However, the mean weight for this group was disproportianately affected by low foetal body weights in a single litter in this group. There did not appear to be a generalised impact on the entire group. In this case, the observations indicate no clear treatment-related foetal toxicity.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No treatment-related effect on the sex ratio of foetuses was noted at any dose compared to the vehicle controls.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There were no statistically significant differences in either the mean litter size or the number of live foetuses per doe across dose groups when compared to the vehicle control.

External malformations:

no effects observed

Description (incidence and severity):

No external abnormalities were noted during gross examinations of foetuses at any dose.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The noted skeletal anomolies in the study are common findings for foetuses for this species and strain. The morphologic observations did not occur in a dose-dependent pattern, nor was the severity linked to an increase in dose. The incidences of absent sternum No. 5, a measure of delayed ossification were found to be higher in controls compared to any other treatment group. Other indicators of delayed ossification such as the absence of proximal phalanges, were found to be single incidences within a litter both in the control group and treatment groups. Poor ossification in sternum No. 5 and No. 6, an indicator of delayed ossification, wsa the only variation to be found to occur as multiple incidences within a litter. This variation however is considered to be less significant than the absence of sternum No. 5. The conflicting results indicate that these findings are incidental.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related abnormalities were observed during visceral examinations of foetuses at any dose. The noted findings [pale coloured kidneys and dilated renal pelvis] are common findings for foetuses of this species and strain. The observations were not dose dependent, nor was the severity of the anomaly increased with dose. This result supports the conclusion that the findings are incidental and that the test material did not produce an adverse effect during foetal development of the soft tissues.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

- Crown rump length:
The mean foetal crown-rump length for both sexes was 92.1, 91.1, 89.3 and 82.3 mm for groups G1, G2, G3 and G4, respectively. A statistically significant reduction in the mean foetal crown-rump length was noted in the high dose group when compared to controls. This was -10.7 % relative to the vehicle control suggesting a marginal but biologically relevant effect on foetal maturation which correlated to the degree of skeletal ossification. One litter in this group had low crown-rump lengths which was found to disproportianately affect the mean. There was no apparent trend when considering all the litters of this group. In addition, there were no correlating statistically significant difference for any other parameter. These observations did not indicate a clear treatment-related effect.

Remarks on result:

not determinable due to adverse toxic effects at highest dose / concentration tested

Abnormalities:

not specified

Developmental effects observed:

not specified

Dose Formulation Analysis

The results for chemical homogeneity and dose concentration verification are within the acceptance limits of ± 10 % of the nominal concentrations.

Table 1: Mean Maternal Thymus Weights

Dose Group (mg/kg)	Thymus (g)	Thymus (% of control)	GD 6-29 (% of control)
G1: Control	2.2353	100.0	0.0
G2: 4	2.1214	94.9	-5.1
G3: 20	2.0209	90.4	-9.6
G4: 80	1.9499	87.2	-12.8

 

Table 2: Foetal Data

Dose Group (mg/kg)	Implantation Sites (mean)	Live Foetuses (mean)	Dead Foetuses (Litters)	Pre-implantation Loss (%)	Post-implantation Loss (%)	Early resorptions (mean)	Late resorptions (mean)
G1: Control	5.1	4.9	0 (0)	0.9	3.1	0.2	0.05
G2: 4	6.0	5.7	2 (2)	0.8	3.5	0.2	0.05
G3: 20	5.5	5.0	2 (2)	2.3	6.4	0.4	0.00
G4: 80	5.0	4.6	3 (1)	4.9	5.7	0.3	0.00

 

Table 3: Mean Foetal Body Weights

Dose Group (mg/kg)	Mean weight (g)	Average foetal crown-rump length (mm)
G1: Control	36.6	92.1
G2: 4	37.3	91.1
G3: 20	35.5	89.3
G4: 80	32.3	82.3*

 * P<0.05

Table 4: Summary of foetal skeletal observations (malformations)

Parameter	Group	G1 (control)	G2 (4 mg/kg)	G3 (20 mg/kg)	G4 (80 mg/kg)
	Number of litters	19	21	19	20*
	Numbers of foetuses	93	119	95	91
Sternum
Sternum No. 5 Absent	No.	7 (5)	4 (3)	4 (3)	5 (4)
	%	7.6	3.4	4.2	4.3
Fore limb
Proximal Phalanx No. 1 Absent	No.	2 (2)	1 (1)	1 (1)	5 (5)
	%	2.2	0.8	1.1	5.6
Proximal Phalanx No. 5 Absent	No.	2 (2)	1 (1)	1 (1)	5 (5)
	%	2.2	0.8	1.1	5.6

The number of litters affected are indicated in parentheses

*One doe litter had all dead foetuses

 

Table 5: Summary of foetal skeletal observations (variations)

Parameter	Group	G1 (control)	G2 (4 mg/kg)	G3 (20 mg/kg)	G4 (80 mg/kg)
	Number of litters	19	21	19	20*
	Numbers of foetuses	93	119	95	91
Skull
Parietal bones – poor ossification	No.	0 (0)	1 (1)	0 (0)	3 (3)
	%	0.0	0.8	0.0	2.9
Inter-parietal bones- poor ossification	No.	0 (0)	1 (1)	0 (0)	1 (1)
	%	0.0	0.8	0.0	1.3
Ribs
Rib at Lumbar vertebrae No. 1 – supplementary, bilateral	No.	0 (0)	1 (1)	2 (1)	1 (1)
	%	0.0	0.8	2.1	0.8
Rib at Lumbar vertebrae No. 1 – supplementary, unilateral	No.	0 (0)	2 (2)	0 (0)	0 (0)
	%	0.0	1.7	0.0	0.0
Sternum No. 5 – Poor ossification	No.	4 (3)	1 (1)	4 (3)	10 (6)
	%	4.4	0.8	4.2	8.7
Sternum No. 6 – Poor ossification	No.	4 (3)	1 (1)	3 (3)	10 (6)
	%	5.3	0.8	3.2	8.7

The number of litters affected are indicated in parentheses

*One doe litter had all dead foetuses

Conclusions:

Under the conditions of the study the NOAEL for maternal toxicity was determined to be 20 mg/kg based on a biologically relevant depression in thymus weight at the 80 mg/kg dose. However, no treatment-related effects were noted in any of the developmental toxicity parameters investigated; therefore the NOAEL for developmental toxicity was concluded to be 80 mg/kg, the highest dose tested.

Executive summary:

The purpose of this study was to assess the effects of prenatal exposure of pregnant female New Zealand White rabbits and their developing foetuses to the test material when administered by oral gavage in a peanut oil vehicle at 2 mL/kg to the mated females from gestation day [GD] 6 to 28. In life examinations included checks for mortality and clinical signs of toxicity, body weight measurements and an evaluation of food consumption. All the animals were sacrificed on gestation day 29 by iv injection of sodium thiopentone and subjected to detailed gross pathology; the gross pathology included a determination of thymus weight. The gravid uterus was collected by hysterectomy and foetuses were removed by caesarean section. Foetuses were subjected to external, soft tissue and skeletal examination.

No deaths or abortions were observed during the experimental period and there were no clinical signs recorded which were indicative of overt toxicity and there were no treatment related differences in average feed consumption or body weights observed at any dose. A total number of 19 (79 %), 21 (88 %), 19 (79 %) and 20 (83 %) mated females were confirmed pregnant in groups G1, G2, G3 and G4, respectively. Gross necropsy revealed a treatment-related effect on the thymus. The mean maternal thymus weight was reduced 5.1, 9.6, and 12.8 % in the 4 mg/kg [low], 20 mg/kg [mid], and 80 mg/kg [high] dose groups, respectively. No other gross pathological findings were noted in any dose group.

Investigation of reproduction parameters revealed no treatment related effects in any of the following: mean gravid uterus weight, number of corpora lutea, number of implantation sites, number of early or late resorptions, percentage of pre and post implantation loss, sex ratio, mean litter size, foetal weight or the number of live foetuses per doe across all the groups.

The mean foetal weights [combined sexes] were 36.6, 37.3, 35.5, and 32.3 grams for groups G1, G2, G3 and G4, respectively. Mean foetal weights were not statistically significantly different across the dose groups when compared to controls. However, at the high dose the mean foetal body weight is -11.9 % relative to controls which suggests a biologically-relevant, but marginal effect on foetal maturation. This however, was found to be disproportionately affected by a single litter in this group who had low body weights. The other litters in this group did not follow the same trend.

Foetal crown-rump length was statistically significantly different from the controls in the highest dose group. This result was affected by one litter in this group who had disproportionately low crown-rump lengths when compared to the other litters of this group. In addition, there were no correlating statistically significant differences for any other developmental parameter.

No external abnormalities were noted during gross examinations of foetuses at any dose and no treatment-related abnormalities were observed during visceral examinations of foetuses at any dose.

Therefore, under the conditions of the study the NOAEL for maternal toxicity was determined to be 20 mg/kg based on a biologically relevant depression in thymus weight at the 80 mg/kg dose. However, no treatment-related effects were noted in any of the developmental toxicity parameters investigated; therefore the NOAEL for developmental toxicity was concluded to be 80 mg/kg, the highest dose tested.

Reference 4

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted on read-across material

Justification for type of information:

Read-across to structurally similar substance Dioctyltin bis (2-ethylhexylmercaptoacetate) (DOTE) (CAS No. 15571-58-1), see attached justification.

Reason / purpose for cross-reference:

read-across source

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

organ weights and organ / body weight ratios

other: maternal toxicity

Dose descriptor:

LOAEL

Effect level:

80 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

organ weights and organ / body weight ratios

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

80 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Remarks on result:

not determinable due to adverse toxic effects at highest dose / concentration tested

Abnormalities:

not specified

Developmental effects observed:

not specified

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

15 mg/kg bw/day

Study duration:

subacute

Species:

mouse

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Deficiencies in the results from the the registered substance (DOTI) data mean that the conclusions are to be taken from the two key studies which were conducted on the read across substance (DOTE, CAS 15571-58-1).

Read across to (DOTE) (CAS No 15571 -58 -1).

Key Study 1 - Developmental Toxicity Study in Mice (2014), Study conducted with the read across substance (DOTE, CAS 15571-58-1)

The purpose of this study was to assess the effects of prenatal exposure of pregnant female Swiss mice and their developing foetuses to the test material when administered by oral gavage in a peanut oil vehicle at 5 mL/kg to the mated females from gestation day [GD] 5 to 17. In life examinations included checks for mortality and clinical signs of toxicity, body weight measurements and an evaluation of food consumption. All the animals were sacrificed on gestation day 18 by carbon dioxide exposure and subjected to detailed gross pathology; the gross pathology included a determination of thymus size. The gravid uterus was collected by hysterectomy and foetuses were removed by caesarean section. Foetuses were subjected to external, soft tissue and skeletal examination.

No deaths were observed during the experimental period and there were no clinical signs recorded which were indicative of overt toxicity. There were no statistically significant differences in maternal body weights across the dose groups on any single gestation day. However, there was a clear dose-related pattern of reduced body weights beginning after GD6, the first day of dosing, and continuing for the duration of the study. The high dose was the most severely affected and a dose-related decrement relative to the control body weights can be seen across all doses particularly from GD16 to GD18. At 30 mg/kg the maternal weight effect was marginal, but maternal body weight gain in the 60 mg/kg high dose group was 11.5 [uncorrected] and 26.6 % [corrected] less than the vehicle control, a clear adverse effect. There was a treatment-related macroscopic finding of reduced maternal thymus weight.

The mean maternal thymus weight was statistically significantly reduced in the 30 mg/kg [mid] and 60 mg/kg [high] dose groups. The mean maternal thymus weight in the low dose mice was reduced relative to controls, but was not a statistically significant difference. No other gross pathological findings were noted in any dose group.

No treatment related effects were noted in mean gravid uterus weight, no. of corpora lutea, no. of implantations in all the groups, no. of early or late resorptions and percentage of post implantation loss.

No external abnormalities were noted during gross examinations of foetuses at any dose and no treatment-related abnormalities were observed during visceral examinations of foetuses at any dose.

Therefore, under the conditions of the study the NOAEL for maternal toxicity was determined to be 15 mg/kg based on a biologically relevant depression in thymus size at the 30 mg/kg dose. However, no treatment-related effects were noted in any of the developmental toxicity parameters investigated; therefore the NOAEL for developmental toxicity was concluded to be 60 mg/kg, the highest dose tested.

Key Study 2 - Developmental Toxicity Study in Rabbits (2014) Study conducted with the read across substance (DOTE, CAS 15571-58-1)

The purpose of this study was to assess the effects of prenatal exposure of pregnant female New Zealand White rabbits and their developing foetuses to the test material when administered by oral gavage in a peanut oil vehicle at 2 mL/kg to the mated females from gestation day [GD] 6 to 28. In life examinations included checks for mortality and clinical signs of toxicity, body weight measurements and an evaluation of food consumption. All the animals were sacrificed on gestation day 29 by iv injection of sodium thiopentone and subjected to detailed gross pathology; the gross pathology included a determination of thymus weight. The gravid uterus was collected by hysterectomy and foetuses were removed by caesarean section. Foetuses were subjected to external, soft tissue and skeletal examination.

No deaths or abortions were observed during the experimental period and there were no clinical signs recorded which were indicative of overt toxicity and there were no treatment related differences in average feed consumption or body weights observed at any dose. A total number of 19 (79 %), 21 (88 %), 19 (79 %) and 20 (83 %) mated females were confirmed pregnant in groups G1, G2, G3 and G4, respectively. Gross necropsy revealed a treatment-related effect on the thymus. The mean maternal thymus weight was reduced 5.1, 9.6, and 12.8 % in the 4 mg/kg [low], 20 mg/kg [mid], and 80 mg/kg [high] dose groups, respectively. No other gross pathological findings were noted in any dose group.

Investigation of reproduction parameters revealed no treatment related effects in any of the following: mean gravid uterus weight, number of corpora lutea, number of implantation sites, number of early or late resorptions, percentage of pre and post implantation loss, sex ratio, mean litter size, foetal weight or the number of live foetuses per doe across all the groups.

The mean foetal weights [combined sexes] were 36.6, 37.3, 35.5, and 32.3 grams for groups G1, G2, G3 and G4, respectively. Mean foetal weights were not statistically significantly different across the dose groups when compared to controls. However, at the high dose the mean foetal body weight is -11.9 % relative to controls which suggests a biologically-relevant, but marginal effect on foetal maturation. This however, was found to be disproportionately affected by a single litter in this group who had low body weights. The other litters in this group did not follow the same trend.

Foetal crown-rump length was statistically significantly different from the controls in the highest dose group. This result was affected by one litter in this group who had disproportionately low crown-rump lengths when compared to the other litters of this group. In addition, there were no correlating statistically significant differences for any other developmental parameter.

No external abnormalities were noted during gross examinations of foetuses at any dose and no treatment-related abnormalities were observed during visceral examinations of foetuses at any dose.

Therefore, under the conditions of the study the NOAEL for maternal toxicity was determined to be 20 mg/kg based on a biologically relevant depression in thymus weight at the 80 mg/kg dose. However, no treatment-related effects were noted in any of the developmental toxicity parameters investigated; therefore the NOAEL for developmental toxicity was concluded to be 80 mg/kg, the highest dose tested.

Supporting information is available from three studies conducted with mixtures containing the registered substance as the main constituent:

Developmental Toxicity Study in Rabbits, 1992 (Study conducted on mixture of 80 % DOTI, CAS 26401-97-8: 20 % MOTI, CAS 26401-86-5)

The teratogenicity of the test material was investigated in a study similar to OECD 414 using New Zealand white rabbits. The study was awarded a reliability score of 3 in accordance with the criteria set forth by Klimisch et al. (1997).

Female rabbits were treated once a day by gavage from day 6 to day 18 of gestation inclusively at 1, 10 and 100 mg/kg/day. The selection of dosages was based on a preliminary embryotoxicity study that had already been performed. The dams were sacrificed on day 28 p.c. and were submitted to gross necropsy including the examination of the foetuses. The effects of the test material were assessed on the basis of clinical observations, body weight and reproduction data of parental females and the examination of foetuses.

At the 1 mg/kg/day treatment level there were no test material related effects. At 10 mg/kg/day there was a slight increase (p > 0.05) in minor skeletal head anomalies (incompletely ossified bones of the skull). At 100 mg/kg/day there was an increased incidence of abortions (p < 0.05); a slight increase (p < 0.05) in postimplantational loss (mainly early resorptions); a moderate foetal weight reduction (p < 0.05); a slight increase (p < 0.05) in minor visceral anomalies (severely dilated renal pelves and additional small vessels originating from the aortic arch); a marked increase (p < 0.05) in minor skeletal head anomalies (incompletely ossified bones of the skull) and a moderate increase (p < 0.05) in skeletal variations of the sternum and feet bones (not or incompletely ossified sternebrae and feet bones).

The statistically non-significant slight increase in minor skeletal head anomalies at the intermediate dose level could indicate a marginal retardative effect on foetal development. Clear-cut effects were found at the high dose level. The comparably high number of four abortions was considered to result at least partly from a slight maternal toxic effect. With regard to embryolethality, slight effects were revealed by the increase in post-implantational loss and the consequently reduced number of foetuses per dam. The foetal weight reduction together with the increase in minor anomalies and variations indicated a test material-related marked retardative effect on foetal development.

Though a slightly higher incidence of foetuses with major anomalies was observed at the high dose level (4 foetuses in contrast to one foetus in each of the remaining groups) the lack of a statistically significant difference to the control group as well as the inconsistency with regard to the type of anomalies did not point towards a compound-related effect. Even if this finding was regarded as an indication, of a weak teratogenic effect of the high dose this would have no further implications for risk estimation since this dose level had already been classified as a clear-cut embryotoxic dose.

Under the conditions of this study no substance-related effects were observed at the low dose of 1 mg/kg/day in rabbits whereas only a marginal retardative effect on foetal development could be attributed to treatment with the intermediate dose of 10 mg/kg/day. At the high dose level of 100 mg/kg/day, clear-cut embryotoxic effects, i.e. an increased rate of abortions and embryolethal effects as well as marked retardations of foetal development, were induced by the test material.

Developmental Toxicity study in Rats, 1991 (Study conducted on mixture of 80 % DOTI, CAS 26401-97-8: 20 % MOTI, CAS 26401-86-5)

The teratogenicity of the test material was investigated in a study similar to OECD 414 using Han-Wistar rats. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

Four groups of 25 inseminated female rats eachwere dosed intragastrically from day 6 through 15 of gestation with oily solutions of the test material at 1, 5 or 25 mg/kg/day or the vehicle (control group). All dams were sacrificed on day 21 p.c. and were submitted to gross necropsy including the examination of the foetuses. The effects of the test material were assessed on the basis of clinical observations, body weight and reproduction data of parental females as well as examination of foetuses.

Up to the intermediate dose of 5 mg/kg/day no test material-related effects were seen. At the high dose level of 25 mg/kg/day a slight, statistically not significant decrease in corrected body weight and corrected body weight gain of the dams (day 6 to 21 p.c.) and a slight increase in the percentage of dead foetuses (p< 0.05) were observed.

The decreased corrected body weight of the dams on day 21 p.c. in combinationwith the reduced corrected body weight gain indicated a marginal maternal toxic effect of the test material.

Though due to one single animal, the increase in the incidence of dead foetuses at the high dose level was considered to result from a marginal embryo-fetolethal effect. Since the results of the already mentioned preliminary study clearly demonstrated a moderate embryolethal effect after a dose of 30 mg/kg/d, the actual high dose of 25 mg/kg/d had to be regarded as the threshold dose with regard to embryolethality.

Under the conditions of this study, no adverse effects were observed up to the intermediate dose of 5 mg/kg/d whereas the high dose of 25 mg/kg/d produced marginal maternal toxic and embryo-foetolethal effects. The latter dose was considered to represent the threshold dose with regard to embryo-lethality.

The RAC position (2011) was that there was only weak evidence on developmental effects seen from this rat study since dead foetuses were seen only from one dam.

Thus no adverse effects which could be attributed to developmental toxicity are found.

Developmental Study in Mice, 2001 (Study conducted on mixture of 80 % DOTI, CAS 26401-97-8: 20 % MOTI, CAS 26401-86-5)

The developmental toxicity of the test material was investigated in NMRI mice. During the study, dams were treated orally with doses of test material at 20, 30, 45, 67, or 100 mg/kg/day from gestation day 6 through 17 (plug 5 day 1). The animals were observed for signs of toxicity. Body weight was measured on day 1 of pregnancy, then every day throughout the treatment period. At necropsy, the thymus and liver weights of the dams were recorded and the ovaries and uterine content was examined.  All viable foetuses were weighed and carefully inspected macroscopically for external structural anomalies. All litters per dose group were used for evaluation of skeletal anomalies.

Under the conditions of the study, resorption rates were significantly increased and foetal weights significantly reduced in the study group at the 2 highest doses. External anomalies, such as bent forelimbs, cleft palate, and exencephaly were reported in the group treated with 100 mg/kg/day, with the 67-mg/kg/d dose also exhibiting a significant increase in cleft palate. Moreover, an increase in skeletal anomalies was reported in foetuses exposed to 100 mg/kg/day. The doses of 20, 30, and 45 mg/kg/day elicited a significant increase in supernumerary lumbar ribs.

The test material was therefore concluded to be embryo-foetotoxic and concluded to induce developmental effects.

The study failed to fulfil the requirements of a GLP OECD 414 guideline study since detailed information on the following were not included: maternal body weight, food consumption, housing, dosing, analysis of the dosing formulations or individual animal data. Furthermore, no information was given on foetal sex ratio, foetal weight per sex or internal malformations and no comparison to historical control data was carried out. The study was therefore considered to be deficient and the results are not taken forward for hazard classification.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008 (CLP), the substance does not require classification with respect to either reproductive or developmental toxicity.