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

Toxicity to reproduction

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1996

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

247-666-0

EC Name:

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

Cas Number:

26401-97-8

Molecular formula:

C36-H72-O4-S2-Sn

IUPAC Name:

6-methylheptyl 14-methyl-4,4-dioctyl-7-oxo-8-oxa-3,5-dithia-4-stannapentadecan-1-oate

Constituent 2

Reference substance name:

Triisooctyl 2,2',2''-[(octylstannylidyne)tris(thio)]triacetate

EC Number:

247-665-5

EC Name:

Triisooctyl 2,2',2''-[(octylstannylidyne)tris(thio)]triacetate

Cas Number:

26401-86-5

Molecular formula:

C38H74O6S3Sn

IUPAC Name:

Triisooctyl 2,2',2''-[(octylstannylidyne)tris(thio)]triacetate

Test material form:

liquid

Details on test material:

- Storage conditions: at room temperature, in a closed container, protected from light and humidity

Test animals

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.

Administration / exposure

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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

20 ppm

Doses / concentrations 2

Dose / conc.:

60 ppm

Doses / concentrations 3

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).

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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.

Effect levels (P0)

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

Results: F1 generation

General toxicity (F1)

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.

Developmental neurotoxicity (F1)

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 (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

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

Results: F2 generation

General toxicity (F2)

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

Developmental neurotoxicity (F2)

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 (F2)

Developmental immunotoxicity:

not examined

Overall reproductive toxicity

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

Applicant's summary and conclusion

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).