Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information
Key study: Experimental results: OECD guideline 422. GLP study.
Based on the results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg bw/day was derived.
Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 06 December 2011 to 02 April 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Age at start F0-treatment: Approximately 10 weeks.
- Weight at study initiation: Males: Mean: 284-286 g. Females: Mean: 196-197 g
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females
were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
Lactation: Pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm). During locomotor activity monitoring of the dams the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left
without their dam or a bottle filled with warm water for longer than 30-40 minutes.
General: Sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment/nesting material (Enviro-dri,
Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment or bedding material.
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water (e.g. ad libitum): Free access to tap-water.
- Acclimation period: Acclimatization F0: At least 5 days prior to start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24 ºC
- Humidity (%): 40 to 70%
- Air changes (per hr): approximately 15 room air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle.

IN-LIFE DATES: From: 06 December 2011 To: 02 April 2012
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for
density (1.084 g/cm3) of the test substance.

Details on mating procedure:
- M/F ratio per cage: Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating occurred, the males and females were separated.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations analysed in the formulations at dose concentrations of 10, 30, 100 and 200 mg/ml were in agreement with target concentrations
(i.e. mean accuracies between 85% and 115%).

The formulations at the entire range were homogeneous (i.e. coefficient of variation ≤ 10%).

Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 6 hours.
Duration of treatment / exposure:
Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy.

Females were exposed for 41-54 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy).
Frequency of treatment:
Once daily for 7 days per week.
Remarks:
Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10 animals/sex/dose
Control animals:
yes
Details on study design:
- Dose selection rationale: Based on the results of a 10-day dose range finding study, the dose levels for this combined 28-day oral gavage study with reproduction/developmental toxicity screening test were selected to be 100, 300 and 1000 mg/kg bw/day.
Parental animals: Observations and examinations:
MORTALITY / VIABILITY: Yes
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards, detailed clinical observations were made in all animals. Once prior to start of
treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed
on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Weekly, except for males and females which were housed together for mating and for females without evidence of
mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 postcoitum and on Days 1 and 4 of lactation.

OTHER:
General reproduction data:
Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of pregnant females was examined to detect signs of abortion or premature birth. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.
Sperm parameters (parental animals):
Parameters examined in male parental generations:
Organ weights: testis weight, epididymis weight.

From the selected 5 males of the control and high dose group, and all males suspected to be infertile, additional slides of the testes were prepared to examine staging of spermatogenesis. The testes were processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin.
Litter observations:
PARAMETERS EXAMINED
Each litter was examined to determine the following, if practically possible:
Mortality / Viability: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined.
Clinical signs: At least once daily, detailed clinical observations were made for all animals.
Body weights: Live pups were weighed on Days 1 and 4 of lactation.
Sex: Sex was determined for all pups on Days 1 and 4 of lactation.

Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
Necropsy was conducted on the following days:
Females which delivered: Lactation Days 5-7.
Females which failed to deliver: Post-coitum Days 26-27 (females with evidence of mating) or approximately 21 days after the last day of the mating
period (females without evidence of mating).
Males: Following completion of the mating period (a minimum of 28 days of dose administration).

All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid the reproductive organs. The numbers of former implantation sites and corpora lutea were recorded for all paired females.

HISTOPATHOLOGY: Yes
Samples of the following tissues and organs were collected and fixed in 10% buffered formalin.
Selected 5 animals/sex/group:
Identification marks: not processed
Ovaries
Adrenal glands
(Pancreas)
(Aorta)
Peyer's patches [jejunum, ileum] if detectable
Brain - cerebellum, mid-brain, cortex
Pituitary gland
Caecum
Preputial gland
Cervix
Prostate gland
Clitoral gland
Rectum
Colon
(Salivary glands - mandibular, sublingual)
Coagulation gland
Sciatic nerve
Duodenum
Seminal vesicles
Epididymides
Skeletal muscle
Eyes (with optic nerve (if detectable) and Harderian gland)
(Skin)
Spinal cord -cervical, midthoracic, lumbar
Female mammary gland area
Spleen
Femur including joint
Sternum with bone marrow
Heart
Stomach
Ileum
Testes
Jejunum
Thymus
Kidneys
Thyroid including parathyroid if detectable
(Lacrimal gland, exorbital)
(Tongue)
(Larynx)
Trachea
Liver
Urinary bladder
Lung, infused with formalin
Uterus
Lymph nodes - mandibular, mesenteric
Vagina
(Nasopharynx)
All gross lesions
(Esophagus)

All remaining animals, females which failed to deliver:
Cervix
Preputial gland
Clitoral gland
Prostate gland
Coagulation gland
Seminal vesicles
Epididymides
Testes
Mammary gland area
Uterus
Ovaries
Vagina
Identification marks: not processed
All gross lesions

The following slides were examined by a pathologist:
- The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4.
- The additional slides of the testes of the selected 5 males of Groups 1 and 4 and all males suspected to be infertile to examine staging of spermatogenesis.
- All gross lesions of all animals (all dose groups).
- The reproductive organs of all males that failed to sire and all females that failed to deliver healthy pups.

ORGAN WEIGHTS:
The following organ weights and terminal body weight were recorded from the following animals on the scheduled day of necropsy:
Selected 5 animals/sex/group:
Adrenal glands
Spleen
Brain
Testes
Epididymides
Thymus
Heart
Uterus (including cervix)
Kidneys
Prostate
Liver
Seminal vesicles including coagulating glands
Ovaries
Thyroid including parathyroid

All remaining males:
Epididymides
Testes
Postmortem examinations (offspring):
GROSS NECROPSY
Pups surviving to planned termination were killed by decapitation on Days 5-7 of lactation.

All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated.
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.

The following additional methods of statistical analysis were used:
Locomotor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences followed by the Wilcoxon test to compare the treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Reproductive indices:
Mating index (%), Fertility index (%), Conception index (%), Gestation index (%), Duration of gestation.
Offspring viability indices:
Percentage live males at First Litter Check, Percentage live females at First Litter Check, Percentage of postnatal loss Days 0-4 of lactation, Viability index.
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY
No mortality occurred during the study period.

No clinical signs of toxicity were noted during the observation period. Incidental findings that were noted for control and/or treated animals included
alopecia and scabbing on different parts of the body. These findings occurred within the range of background findings to be expected for rats of this
age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological relevance.

BODY WEIGHT AND WEIGHT GAIN
Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.

FOOD CONSUMPTION AND COMPOUND INTAKE
Food consumption before or after allowance for body weight was similar between treated and control animals.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Staging of spermatogenesis did not provide any evidence of test article related impairment to the spermatogenetic cycle.

REPRODUCTIVE DATA
The mating, fertility and conception indices, precoital time, and numbers of corpora lutea and implantation sites were considered to be unaffected by treatment up to 1000 mg/kg bw/day.

There were 7, 8, 10 and 10 litters available for evaluation in the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. In the control group, one female was not mated and two other females were not pregnant. At 100 mg/kg, one female was not mated and another female was not pregnant. In the absence of a treatment-related distribution, these findings were not considered toxicologically relevant.

ORGAN WEIGHTS
There were no toxicologically relevant changes in organ weights or organ to body weight ratios seen with treatment up to 1000 mg/kg.

Statistically significant changes included lower seminal vesicle organ weights (absolute and relative to body weight) for males at 100, 300 and 1000
mg/kg bw/day, higher relative brain weights for males at 300 mg/kg bw/day and higher relative adrenal weights for males at 1000 mg/kg bw/day. These findings were considered not to be toxicologically relevant as changes were very slight, a treatment-related trend was absent, no corroborative
histopathological changes were evident and/or the reproductive ability of the males was not affected.

GROSS PATHOLOGY
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.

Incidental findings seen for control and/or treated animals included pelvic dilation of the kidneys (unilateral), greenish, soft nodule on the tail of the
epididymides (both unilateral and bilateral), redbrown discoloration of the thymus, enlarged mandibular lymph nodes (unilateral), tan discoloration of
the clitoral glands, and alopecia on different sides of the body. The incidence of these macroscopic findings was within the background range of
findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. They were therefore considered to be of no toxicological relevance.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no microscopic observations attributed to the test article.
No significant abnormalities were seen in the reproductive organs of the paired animals that failed to conceive.

GESTATION
Gestation index and duration of gestation were in the same range for treated groups and controls.

PARTURITION / MATERNAL CARE
No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth.
No deficiencies in maternal care were observed.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed at the highest dose level.
Critical effects observed:
no
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
VIABILITY (OFFSPRING)
The number of pups found dead or went missing during the first days of lactation were 1, 1, 3 and 1 in the control, 100, 300 and 1000 mg/kg bw/day groups. Pups that went missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

CLINICAL SIGNS (OFFSPRING)
There were no clinical signs noted.

BODY WEIGHT (OFFSPRING)
Body weights of pups were considered to have been unaffected by treatment.

SEXUAL MATURATION (OFFSPRING)
Number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings.

GROSS PATHOLOGY (OFFSPRING)
Incidental macroscopic findings of pups that were found dead included no milk in the stomach and partial cannibalism. There were no macroscopic findings noted among surviving pups. The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed at the highest dose level.
Reproductive effects observed:
no
Conclusions:
Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg bw/day was derived.
Executive summary:

The test substance was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 100, 300, or 1000 mg/kg bw/day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 41- 54 days). Formulation analysis showed that the formulations were prepared accurately and homogenously, and were stable for at least 6 hours at room temperature.

Parental results: No parental toxicity was evident up to the highest dose level tested (1000 mg/kg bw/day). The statistically significantly higher locomotor activity (total movements and ambulations) noted for females at 1000 mg/kg bw/day as compared to controls was not considered adverse, since changes were relatively slight, and occurred in the absence of concurrent clinical signs or changes in other functional observation tests. No treatment-related effects were noted on any of the remaining parental parameters investigated in this study (i.e. clinical appearance, hearing ability, pupillary reflex, static righting reflex and grip strength, body weight, food consumption, clinical laboratory investigations, macroscopic examination, organ weights, and microscopic examination).

Reproductive results: No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg bw/day). No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites).

Developmental results: No developmental toxicity was observed up to the highest dose level tested 1000 mg/kg bw/day). No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight and macroscopy).

Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg bw/day was derived.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Klimisch 1 and the study was carried out in accordance with internationally valid GLP principles.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Key study: Experimental results: OECD guideline 422. GLP study.

 

Parental results: No parental toxicity was evident up to the highest dose level tested (1000 mg/kg bw/day). The statistically significantly higher locomotor activity (total movements and ambulations) noted for females at 1000 mg/kg bw/day as compared to controls was not considered adverse, since changes were relatively slight, and occurred in the absence of concurrent clinical signs or changes in other functional observation tests. No treatment-related effects were noted on any of the remaining parental parameters investigated in this study (i.e. clinical appearance, hearing ability, pupillary reflex, static righting reflex and grip strength, body weight, food consumption, clinical laboratory investigations, macroscopic examination, organ weights, and microscopic examination).

Reproductive results: No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg bw/day). No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites).

Developmental results: No developmental toxicity was observed up to the highest dose level tested 1000 mg/kg bw/day). No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight and macroscopy).

Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg bw/day was derived.

Effects on developmental toxicity

Description of key information

Key study: Experimental results: OECD guideline 422. GLP study.

Based on the results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg bw/day was derived.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 06 December 2011 to 02 April 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline 422. GLP study.
Qualifier:
according to
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Age at start F0-treatment: Approximately 10 weeks.
- Weight at study initiation: Males: Mean: 284-286 g. Females: Mean: 196-197 g
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females
were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
Lactation: Pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm). During locomotor activity monitoring of the dams the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left
without their dam or a bottle filled with warm water for longer than 30-40 minutes.
General: Sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment/nesting material (Enviro-dri,
Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment or bedding material.
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water (e.g. ad libitum): Free access to tap-water.
- Acclimation period: Acclimatization F0: At least 5 days prior to start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24 ºC
- Humidity (%): 40 to 70%
- Air changes (per hr): approximately 15 room air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle.

IN-LIFE DATES: From: 06 December 2011 To: 02 April 2012
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for
density (1.084 g/cm3) of the test substance.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations analysed in the formulations at dose concentrations of 10, 30, 100 and 200 mg/ml were in agreement with target concentrations
(i.e. mean accuracies between 85% and 115%).

The formulations at the entire range were homogeneous (i.e. coefficient of variation ≤ 10%).

Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 6 hours.
Details on mating procedure:
- M/F ratio per cage: Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating occurred, the males and females were separated.
Duration of treatment / exposure:
Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy.

Females were exposed for 41-54 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy).
Frequency of treatment:
Once daily for 7 days per week.
No. of animals per sex per dose:
10 animals/sex/dose
Control animals:
yes
Details on study design:
- Dose selection rationale: Based on the results of a 10-day dose range finding study, the dose levels for this combined 28-day oral gavage study with reproduction/developmental toxicity screening test were selected to be 100, 300 and 1000 mg/kg bw/day.
Maternal examinations:
MORTALITY / VIABILITY: Yes
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards, detailed clinical observations were made in all animals. Once prior to start of
treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed
on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Weekly, except for males and females which were housed together for mating and for females without evidence of
mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 postcoitum and on Days 1 and 4 of lactation.

OTHER:
General reproduction data:
Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of pregnant females was examined to detect signs of abortion or premature birth. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.
Fetal examinations:
PARAMETERS EXAMINED
Each litter was examined to determine the following, if practically possible:
Mortality / Viability: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined.
Clinical signs: At least once daily, detailed clinical observations were made for all animals.
Body weights: Live pups were weighed on Days 1 and 4 of lactation.
Sex: Sex was determined for all pups on Days 1 and 4 of lactation.

Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.

The following additional methods of statistical analysis were used:
Locomotor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences followed by the Wilcoxon test to compare the treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Indices:
Mating index (%), Fertility index (%), Conception index (%), Gestation index (%), Duration of gestation.
Percentage live males at First Litter Check, Percentage live females at First Litter Check, Percentage of postnatal loss Days 0-4 of lactation, Viability index.
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY
No mortality occurred during the study period.

No clinical signs of toxicity were noted during the observation period. Incidental findings that were noted for control and/or treated animals included
alopecia and scabbing on different parts of the body. These findings occurred within the range of background findings to be expected for rats of this
age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological relevance.

BODY WEIGHT AND WEIGHT GAIN
Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.

FOOD CONSUMPTION AND COMPOUND INTAKE
Food consumption before or after allowance for body weight was similar between treated and control animals.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Staging of spermatogenesis did not provide any evidence of test article related impairment to the spermatogenetic cycle.

REPRODUCTIVE DATA
The mating, fertility and conception indices, precoital time, and numbers of corpora lutea and implantation sites were considered to be unaffected by treatment up to 1000 mg/kg bw/day.

There were 7, 8, 10 and 10 litters available for evaluation in the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. In the control group, one female was not mated and two other females were not pregnant. At 100 mg/kg, one female was not mated and another female was not pregnant. In the absence of a treatment-related distribution, these findings were not considered toxicologically relevant.

ORGAN WEIGHTS
There were no toxicologically relevant changes in organ weights or organ to body weight ratios seen with treatment up to 1000 mg/kg.

Statistically significant changes included lower seminal vesicle organ weights (absolute and relative to body weight) for males at 100, 300 and 1000
mg/kg bw/day, higher relative brain weights for males at 300 mg/kg bw/day and higher relative adrenal weights for males at 1000 mg/kg bw/day. These findings were considered not to be toxicologically relevant as changes were very slight, a treatment-related trend was absent, no corroborative
histopathological changes were evident and/or the reproductive ability of the males was not affected.

GROSS PATHOLOGY
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.

Incidental findings seen for control and/or treated animals included pelvic dilation of the kidneys (unilateral), greenish, soft nodule on the tail of the
epididymides (both unilateral and bilateral), redbrown discoloration of the thymus, enlarged mandibular lymph nodes (unilateral), tan discoloration of
the clitoral glands, and alopecia on different sides of the body. The incidence of these macroscopic findings was within the background range of
findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. They were therefore considered to be of no toxicological relevance.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no microscopic observations attributed to the test article.
No significant abnormalities were seen in the reproductive organs of the paired animals that failed to conceive.

GESTATION
Gestation index and duration of gestation were in the same range for treated groups and controls.

PARTURITION / MATERNAL CARE
No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth.
No deficiencies in maternal care were observed.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Abnormalities:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
VIABILITY (OFFSPRING)
The number of pups found dead or went missing during the first days of lactation were 1, 1, 3 and 1 in the control, 100, 300 and 1000 mg/kg bw/day groups. Pups that went missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

CLINICAL SIGNS (OFFSPRING)
There were no clinical signs noted.

BODY WEIGHT (OFFSPRING)
Body weights of pups were considered to have been unaffected by treatment.

SEXUAL MATURATION (OFFSPRING)
Number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings.

GROSS PATHOLOGY (OFFSPRING)
Incidental macroscopic findings of pups that were found dead included no milk in the stomach and partial cannibalism. There were no macroscopic findings noted among surviving pups. The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.
Remarks on result:
not measured/tested
Abnormalities:
not examined
Developmental effects observed:
no
Conclusions:
Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg bw/day was derived.
Executive summary:

The test substance was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 100, 300, or 1000 mg/kg bw/day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 41- 54 days). Formulation analysis showed that the formulations were prepared accurately and homogenously, and were stable for at least 6 hours at room temperature.

Parental results: No parental toxicity was evident up to the highest dose level tested (1000 mg/kg bw/day). The statistically significantly higher locomotor activity (total movements and ambulations) noted for females at 1000 mg/kg bw/day as compared to controls was not considered adverse, since changes were relatively slight, and occurred in the absence of concurrent clinical signs or changes in other functional observation tests. No treatment-related effects were noted on any of the remaining parental parameters investigated in this study (i.e. clinical appearance, hearing ability, pupillary reflex, static righting reflex and grip strength, body weight, food consumption, clinical laboratory investigations, macroscopic examination, organ weights, and microscopic examination).

Reproductive results: No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg bw/day). No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites).

Developmental results: No developmental toxicity was observed up to the highest dose level tested 1000 mg/kg bw/day). No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight and macroscopy).

Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg bw/day was derived.

Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further details are given in the endpoint summary "Toxicity to reproduction"

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar toxicological properties because
-      they are manufactured from similar resp. identical precursors
-      they share structuralsimilarities with common functional groups:glycerol as head-group, PEG-linker and fatty acids
Thus a common mode of action can be assumed. Therefore, read-across from the existing prenatal developmental toxicity studies conducted with the source substances PEG-40 Castor Oil, PEG-40 Hydrogenated Castor Oil and PEG-8 caprylic/capric glycerides is considered as an appropriate adaptation to the standard information requirements of the REACH Regulation for the target substance, in accordance with the provisions of Annex XI, 1.5 of the REACH Regulation.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Further details are given in the endpoint summary "Toxicity to reproduction"

3. ANALOGUE APPROACH JUSTIFICATION
Further details are given in the endpoint summary "Toxicity to reproduction"

4. DATA MATRIX
Further details are given in the endpoint summary "Toxicity to reproduction"
Reason / purpose:
read-across: supporting information
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Species:
other: rat and mouse
Details on maternal toxic effects:
Maternal toxic effects:no effects
Dose descriptor:
NOEL
Remarks:
rat
Effect level:
100 000 ppm
Based on:
act. ingr.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOEL
Remarks:
mouse
Effect level:
10 000 ppm
Based on:
act. ingr.
Basis for effect level:
other: maternal toxicity
Abnormalities:
no effects observed
Dose descriptor:
NOEL
Remarks:
rat
Effect level:
50 000 ppm
Based on:
act. ingr.
Basis for effect level:
other: fetotoxicity
Dose descriptor:
NOEL
Remarks:
mouse
Effect level:
10 000 ppm
Based on:
act. ingr.
Basis for effect level:
other: fetotoxicity
Abnormalities:
no effects observed
Developmental effects observed:
no
Conclusions:
It is concluded that the target substance Reaction products of tryglycerides, C8-C18 (even numbered) and C18-unsaturated, glycerine and ethylene oxide does not have effects on prenatal development based on the results obtained with the structurally related source substancesPEG-40 Hydrogenated Castor Oil and PEG-8 caprylic/capric glycerides.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Klimisch 1 and the study was carried out in accordance with internationally valid GLP principles.
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

Key study: Experimental results: OECD guideline 422. GLP study.

In a study according to OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test), the test substanceReaction products of tryglycerides, C8-C18 (even numbered) and C18-unsaturated, glycerine and ethylene oxidewas administered by daily oral gavage to male and female Wistar Han rats at dose levels of 100, 300, or 1000 mg/kg bw/day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 41- 54 days). Formulation analysis showed that the formulations were prepared accurately and homogenously, and were stable for at least 6 hours at room temperature.

 

Parental results: No parental toxicity was evident up to the highest dose level tested (1000 mg/kg bw/day). The statistically significantly higher locomotor activity (total movements and ambulations) noted for females at 1000 mg/kg bw/day as compared to controls was not considered adverse, since changes were relatively slight, and occurred in the absence of concurrent clinical signs or changes in other functional observation tests. No treatment-related effects were noted on any of the remaining parental parameters investigated in this study (i.e. clinical appearance, hearing ability, pupillary reflex, static righting reflex and grip strength, body weight, food consumption, clinical laboratory investigations, macroscopic examination, organ weights, and microscopic examination).

 

Reproductive results: No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg bw/day). No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites).

 

Developmental results: No developmental toxicity was observed up to the highest dose level tested 1000 mg/kg bw/day). No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight and macroscopy).

 

Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg bw/day was derived.

 

Adaptation of information requirements

According to Annex XI, 1.2 of the REACH regulation some substances may be excluded from testing for prenatal developmental toxicity if it does not appear scientifically necessary. Following the approach outline in guidance on information requirements R7a, integrated test strategy, testing of prenatal developmental toxicity withReaction products oft tryglycerides, C8-C18 (even numbered) and C18-unsaturated, glycerine and ethylene oxide does not appear scientifically necessary, based on the following reasons considered in a weight of evidence approach:

For Reaction products of tryglycerides, C8-C18 (even numbered) and C18-unsaturated, glycerine and ethylene oxide there is sufficient evidence from several independent sources of information leading to the assumption/conclusion that the substance does not have a particular dangerous property (prenatal developmental toxicity after oral administration). The Weight of Evidence analysis demonstrates that the available information is sufficient for an adequate hazard characterisation and classification and labelling purposes. A CSA is available where it is shown, that the exposure to the substance is adequately controlled.

 

1)        There is convincing and valid data available thatReaction products oftryglycerides, C8-C18 (even numbered) and C18-unsaturated, glycerine and ethylene oxide is unreactive or inert

a.        Regarding the chemical structure of the substance, there are no groups associated with certain reactivity like oxidizing properties, electrophilicity or extreme pH-values. This is supported by negative findings from irritation, sensitization and mutagenicity studies.

 

2)        Reliable data is available to justify that the substance is of low toxicity.

a.        Data from a combined Repeated Dose Toxicity study with the Reproduction/Developmental Toxicity Screening Test according to OECD TG 422 (Klimisch1) are available. The No Observed Adverse Effect Level (NOAEL) for repeated dose toxicity, as well as for reproductive and developmental toxicity was considered to be 1000 mg/kg body weight/day, the highest tested dose and limit dose of guideline. The study is already documented as RSS in the technical dossier.

b.        Additional supporting information on similar substances is available showing no evidence of prenatal developmental toxicity. Details are given below.

 

3)        This pattern is coupled with limited human exposure.

a.        The substance is only handled in industrial or commercial installations using predominantly closed systems.

b.        The vapour pressure is sufficiently low that inhalational exposures are unlikely to be of significance, and human exposure is limited to dusts or aerosols unlikely to be inhalable.

c.        The substance is handled only as preparations at low concentrations.

d.        Concerning consumer uses the substance is predominantly used as an ingredient for cosmetic preparations and this use is outside the scope of REACH.

 

The above mentioned claims are substantiated by relevant data which are included in the dossier. An exposure and risk assessment covering all REACH relevant scenarios is also included demonstrating that there is only limited human exposure.

 

Supporting data from related substances

Further supporting data on prenatal developmental toxicity in rat and mouse are available for the structurally related source substance PEG-40 Hydrogenated Castor Oil:

 

In a prenatal developmental toxicity study PEG-40 Hydrogenated Castor Oil was administered to NMRI mice at dose levels of 5000 or 10000 ppm in diet.

There was no statistically significant evidence of either maternal or fetal toxicity. The few malformations observed among the fetuses of the treated dams were

similar to type and number to those found in the control groups.

The NOEL for maternal and fetal toxicity was 10000 ppm, corresponding to 1000 mg/kg bw/d taking into account typical body weight and feed consumption of mouse.

 

In a prenatal developmental toxicity study PEG-40 Hydrogenated Castor Oil was administered to Sprague-Dawley rats at dose levels of 50,000 and 100,000 ppm in diet.

No evidence of either maternal or fetal toxicity was present. A slight but not statistically significant increase occurred in the number of resorptions in the group treated with 100,000 ppm. The type and number of malformations and anomalies found in the fetuses of the experimental groups were similar to those found among the fetuses from the control groups. The investigators concluded that PEG-40 Hydrogenated Castor Oil was not teratogenic.

The NOEL for maternal was 100,000 ppm, corresponding to 10,000 mg/kg bw/d taking into account typical body weight and feed consumption of rats.

The NOEL for fetal toxicity was 50,000 ppm, corresponding to 5,000 mg/kg bw/d taking into account typical body weight and feed consumption of rats.

 

Further, though very limited information is also available on PEG-8 caprylic/capric glycerides:

A prenatal developmental toxicity study was performed in rats with PEG-8 caprylic/capric glycerides.The animals were dosed with 0, 1000, 2000, or 3000 mg/kg bw/day by gavage on days 6 through 17 of gestation. The maternal NOAEL was 2000 mg/kg bw/day; effects on body weight were reported at 3000 mg/kg bw/day. The embryo/fetal NOAEL was 3000 mg/kg bw/day. No signs of embryotoxicity, fetotoxicity or teratogenicity were noted at any dose level.

 

Justification for read-across

This read-across is based on the hypothesis that source and target substances have similar toxicological properties because

-      they are manufactured from similar resp. identical precursors

-      they share structuralsimilarities with common functional groups:glycerol as head-group, PEG-linker and fatty acids

Thus a common mode of action can be assumed.Therefore, read-across from the existing prenatal developmental toxicity studies conducted with the source substances PEG-40 Hydrogenated Castor Oil and PEG-8 caprylic/capric glycerides is considered as an appropriate adaptation to the standard information requirements of the REACH Regulation for the target substance, in accordance with the provisions of Annex XI, 1.5 of the REACH Regulation.

 

The general structure of the target and source substance is shown below (see attachment).

 

Target substance

Source substance

Source substance

Source substance

Substance name

Reaction products of tryglycerides, C8-C18 (even numbered) and C18-unsaturated, glycerine and ethylene oxide

PEG-40 Castor Oil

PEG-40 Hydrogenated Castor Oil

PEG-8 caprylic/capric glycerides

Number of PEG-units (mean)

2

40

40

8

C-chains

C8: 3-10%

C10: 3-8%

C12: 44-54%

C14: 14-20%

C16: 7-11%

C18: 1-4%

C18’: 5-20%

C18’’: 1-4%

Mainly ricinoleic acid (12-hydroxy-9-cis-octadecenoic acid)

Mainly C18satd.-OH (12-hydroxy-octadecanoic acid)

Mainly C8 + C10

 

Differences in the intrinsic properties of the target and source substances could potentially arise from the following facts:

The source substance PEG-40 Hydrogenated Castor Oil contains longer PEG-chains as linker compared to the target substance which on the one hand increases the molecular weight, but on the other hand makes the source substances more hydrophilic. 

The source substance PEG-40 Hydrogenated Castor Oil contains – in contrast to the target substance – the hydroxy-fatty acid ricinoleic acid (12-hydroxy-9-cis-octadecenoic acid).

 

The source substance PEG-8 caprylic/capric glycerides contains only short fatty acid chains, but slightly longer PEG-linkers compared to the target substance, which is expected to increase hydrophilicity and thereby bioavailability of the source substance.

 

The structural similarities between the substances as presented above support the read-across hypothesis. It is concluded that the target substance Reaction products of tryglycerides, C8-C18 (even numbered) and C18-unsaturated, glycerine and ethylene oxide does not have effects on prenatal development based on the results obtained with the structurally related source substancesPEG-40 Hydrogenated Castor Oil and PEG-8 caprylic/capric glycerides.

Justification for classification or non-classification

Based on the available information, the substance is not classified as toxic for reproduction:

Based on the results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg bw/day was derived.