Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

One K1 study was performed according to OECD 421 guideline: rats were dermally exposed to 10, 50, 100 mg/kg bw/d. No toxic effects were observed: the no observed adverse effect level (NOAEL) is greater than 100 mg/kg/day. Also, in the absence of any impairment of reproductive function, the highest dose tested (100 mg/kg/day) was considered to be the no observed effect level (NOEL) in terms of reproductive function and fertility.

Link to relevant study records

Referenceopen allclose all

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-10-30 to 2010-04-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
There were minor protocol deviations considered to have not affected the outcome of the study
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
minor protocol deviations related to temperature and time of observations. These deviations were considered not to have affected the study integrity.
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 9D416 (source: Sponsor)
- Expiration date of the lot/batch: 27-04-2011

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At ambient temperature and protected from light

OTHER SPECIFICS:
- Composition of test material, percentage of components: Primary amine (>90% of total amine), Total acetylatables (6.5-7.1 meq/g), total amine (6.1-6.6 meq/g), water (0.25 max wt%)
Species:
rat
Strain:
other: Crl:CD (SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Ltd., UK
- Age at study initiation: 9-10 weeks
- Weight at study initiation: 314-358 g (males) and 224-268 g (females)
- Fasting period before study: no fasting
- Housing: Individiually and depending upon the phase of the study, animals were housed in P2000 (pre-pairing) or 2154 (post mating) solid-bottomed cages, or RB3 modified cages with stainless steel grid flooring (during mating). Wood based bedding was used, which was sterilised by autoclaving and changed at least twice a week.
- Use of restrainers for preventing ingestion (if dermal): no, after removal of dressing the exposed area was cleaned to reduce the risk of ingestion
- Diet: Standard rodent diet (SDS VRF1 Certified Diet), ad libitum. The diet contained no added antibiotic, or other chemotherapeutic or prophylactic agent
- Water: Potable water taken from the public supply, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23 °C
- Humidity (%): 40 to 70%
- Photoperiod (hrs dark / hrs light): 12/12 (lights on at 6.00 GMT)
Route of administration:
dermal
Vehicle:
water
Details on exposure:
TEST SITE
- Area of exposure: Dorsum between the limb girdles
- % coverage: approx. 10% of total body
- Type of wrap if used: Gauze patch held in place with cotton wool, Tubigrip bandage and surgical tape (semi-occlusive)
- Time intervals for shavings or clipplings: As required

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Exposed area was cleaned with warm tap water and dabbed dry with disposable paper towel/tissues
- Time after start of exposure: not less than six hours (except for females on Day 21 of mating and Day 2 and 3 of lactation when exposure was 3 hours)

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 3 mL/kg
- Concentration (if solution): 3.3, 16.7 and 33.3 mg/mL
- Constant volume or concentration used: yes

USE OF RESTRAINERS FOR PREVENTING INGESTION: no, info on 'Test animals'
Details on mating procedure:
- M/F ratio per cage: one-to-one basis (1:1)
- Length of cohabitation: 14 days
- Proof of pregnancy: Cages were checked daily for ejected copulation plugs and a vaginal smear was examined on the presence of spermatozoa and the stage of the oestrous cycle. The day on which evidence of mating was found was designated Day 0 of gestation.
- After successful mating each pregnant female was caged (how): Individually in 2154 solid-bottomed cages.
- Any other deviations from standard protocol: Not applicable
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each formulation prepared for administration in the first and last preparations were analysed for achieved concentration of the test substance. Four samples were taken (nominally 1 mL accurately weighed) from all groups; 2 assays from each test group and 1 assay from the control group. The remainder was retained as contingency for analysis if any result required confirmation. The method of analysis was LC-MS and was an adaptation of a method supplied by the Sponsor;
Duration of treatment / exposure:
Males were treated daily 15 days before pairing, throughout pairing until Day 3 after the birth of the F1 generation. Females were treated daily for 15 days before pairing, throughout pairing until Day 22 of gestation with recommencement on Day 2 of lactation until Day 3 after the birth of the F1 generation.
Frequency of treatment:
Daily (7 days per week) at 6 hours per day, except for females on Day 21 of mating and Day 2 and 3 of lactation when exposure was only three hours
Details on study schedule:
Age at mating of the mated animals in the study: 11 to 12 weeks
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
control group
Dose / conc.:
10 mg/kg bw/day (nominal)
Remarks:
low dose group
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
mid dose group
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
high dose group
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose levels of 10,50 and 100 mg/kg/day were selected in conjunction with the Sponsor based on findings from previous toxicology studies (dermal LD50 ca. 600 mg/kg; dermal 28-d NOEL of 100 mg/kg bw/d; dermal 90-d NOEL of 16 mg/kg bw/d, systemic 90-d NOEL of >160 mg/kg bw/d).
- Rationale for animal assignment (if not random): The males and females were divided into bodyweight strata (5 g range) and allocated to treatment groups by selecting animals from each bodyweight range in rotation after any grossly atypical animals had been discarded. This procedure ensured that all groups contained populations of rats with similar initial mean and range of bodyweights, and that discarded surplus animals were amongst those with outlying bodyweights.
- Other: The route of administration was chosen to simulate the conditions of human exposure.
Positive control:
Not used
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

DERMAL OBSERVATIONS: daily, before each administration, according to Draize criteria

FOOD CONSUMPTION: mean weekly consumption per animal until mating, mean daily consumption per female animal after mating
Oestrous cyclicity (parental animals):
For 15 days before pairing, daily vaginal smears were taken from all females, using cotton swabs moistened with saline. The smears were subsequently examined to establish the duration and regularity of the oestrous cycle. After pairing with the male, smearing was continued using pipette lavage, until evidence of mating was observed
Sperm parameters (parental animals):
For the assessment of the testes at necropsy, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage-specificity of testicular findings was noted.
Litter observations:
All litters were examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter. Clinical signs, Litter size, sex ratioand body weight of individual offsprings were examined.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: After confirmation that a second mating was not required
- Maternal animals: F0 females were killed on Day 4 of lactation. Females that failed to produce a viable litter were killed on Day 25 after mating. Females whose litter died before Day 4 of lactation were killed on the day the last offspring died.

GROSS NECROPSY
- Gross necropsy consisted of a full macroscopic examination of the tissues; a visual examination of all external features and orifices; an in situ examination of neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera; examination of size and appearance of organs and tissues. For females the number of implantation sites in each uterine horn was counted.

HISTOPATHOLOGY / ORGAN WEIGHTS
Following organs were weighted: epididymides, ovaries, pituitary, prostate, seminal vesicles, testes and uterus with cervix and oviducts. Following tissues were fixed and histologically examined for all adult animals of Groups 1 (control) and 4 (100 mg/kg/day): application site, epididymides, pituitary, ovaries, seminal vesicles, prostate, testes, uterus and vagina. The reproductive organs (i.e mammary area - caudal) for one Group 2 (10 mg/kg/day) female with a litter death were additionally examined.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring sacrificed at 4 days of age.

GROSS NECROPSY
- Gross necropsy consisted of a full macroscopic examination of the tissues; a visual examination of all external features and orifices; an in situ examination of neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera; examination of size and appearance of organs and tissues.
- Premature deaths before weaning were examined similarly as above, if not autolysed or cannibalised. Presence of milk in the stomach was assessed as well.
Statistics:
The following sequence of statistical tests was used for bodyweight and organ weight data: A parametric analysis was performed if Bartlett's test for variance homogeneity was not significant at the 1% level. The F1 approximate test was applied. If the F1 approximate test for monotonicity of dose-response was not significant at the 1% level, Williams' test for a monotonic trend was applied. If the F1 approximate test was significant, suggesting that the dose-response was not monotone, Dunnett's test was performed instead.
A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. The H1 approximate test, the non-parametric equivalent of the F1 test described above, was applied. If the H1 approximate test for monotonicity of dose-response was not significant at the 1% level, Shirley's test for a monotonic trend was applied. If the H1 approximate test was significant, suggesting that the dose-response was not monotone, Steel's test was performed instead.

Significant differences between Control and treated groups were expressed at the 5% (p<0.05) or 1% (p<0.01) level.

For organ weight data, analysis of covariance was performed using terminal bodyweight as covariate
Reproductive indices:
Oestrous cycles
The percentage females showing the following classifications of oestrous cycles before pairing are presented:
- Regular: All observed cycles of 4 or 5 days
- Irregular: At least one cycle of 2, 3 or 6 to 10 days
- Acyclic: At least 10 days without oestrus

Mating performance and fertility
- Percentage mating = (# animals mating/animals paired)*100
- Conception rate (%) = (# animals achieving pregnancy/animals mated)*100
- Fertility index (%) = (# animals achieving pregnancy/animals pairing)*100

Gestation length
- Gestation index (%) = (# live litters born/ # pregnant)*100

Sex ratio in litter
- % males = (# males in litter/ total # offspring in litter)*100
Offspring viability indices:
The following were calculated for each litter:
- Post-implantation survival index (%) = (total # offspring born/ total # uterine implantation sites)*100
Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded.

- Live birth index (%) = (# live offspring on day 1 after littering/ total # offspring born)*100
- Viability index (%) = (# live offspring on day 4 after littering/ #live offspringon day 1 after littering)*100
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
no effects observed
Description (incidence and severity):
Dermal reactions at the application site of male animals were limited to very slight erythema and eschar/scab formation, however there was no evidence for either a treatment related or dose related response in either the number of animals or the number of occasions the signs were observed.
For females before pairing, during gestation and lactation dermal signs at the application site and included very slight/well defined eythema, eschar/scab formation, exfoliation and sloughing. The incidence in terms of the number animals affected and the number of occasions observed was marginally higher at 100 mg/kg/day when compared with Controls, but there was no clear evidence of a dose related response at 10 or 50 mg/kg/day.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Overall mean bodyweight gain for males receiving 100 mg/kg/day was low when compared with Controls, approximately 83% of Controls; however this difference did not attain statistical significance. Bodyweight gain for males at 10 or 50 mg/kg/day was unaffected by treatment.
Bodyweight gain for females during the two week treatment period before being paired for mating was unaffected by treatment with the test substance. During gestation mean bodyweight gain for females receiving 100 mg/kg/day was approximately 88% of controls, however statistical significance was not attained. At 10 or 50 mg/kg/day the bodyweight gain of females during gestation was similar to the Controls.
During lactation the bodyweight gain of females was unaffected by treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not specified
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating performance was unaffected by treatment with all paired animals showing positive evidence of mating. One female at 100 mg/kg/day was not pregnant and because of the small group size this lowered the conception and fertility index at 100 mg/kg/day to 90% compared with 100% in the Control group. However a total of 10 Control females and 10, 10 and 9 females at 10, 50 and 100 mg/kg/day, respectively, achieved pregnancy.
The majority of animals commenced parturition within the normal range of 22 to 23.5 days. At 10 mg/kg/day one female the gestation length was 21.5 days however there was no other evidence for a treatment related reduction in gestation length.
Key result
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
> 100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Key result
Dose descriptor:
NOEL
Remarks:
reproduction and fertility
Effect level:
> 100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Mating performance, fertility and offspring survival and development up to Day4 of age was unaffected by treatment at dose levels up to 100 mg/kg/day.
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Other effects:
not examined
VIABILITY (OFFSPRING)
The mean number of implantations was essentially similar amongst the groups. However at 100 mg/kg/day the mean post-implantation survival and subsequent litter size were low when compared with the Controls; although statistical significance was not attained. Review of the individual litter data revealed that this was attributed to two litters at 100 mg/kg/day, namely 72 and 73. The implantation rate of these animals (nos.72 and 73) was comparable with other animals in the group and although the post-implantation survival rate was low (14 and 35%, respectively), the survival rate in the other seven animals was similar to the Controls ranging between 79 and 100%. Post natal survival for litter nos. 72 and 73 was good and the mean bodyweight of offspring on Day 1 of age was above average, reflecting the low litter size/heavier bodyweight ratio. This heavier weight was maintained to Day 4 of age and there were no significant findings in these dams or offspring at necropsy. It is considered likely that the increased post-implantation loss in these two high dose litters is fortuitous and is exaggerated by the small sample size, and is therefore considered unlikely to be related to treatment. However the potential for higher in-utero loss at 100 mg/kg/day cannot be resolved within the context of the design of this screening study.
Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
> 100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
Key result
Critical effects observed:
no
Reproductive effects observed:
not specified

The mean concentrations in dose formulations analysed during this study were within ± 10% of nominal concentrations, confirming accurate formulation.

Conclusions:
It was concluded that in this study, in terms of general toxicity, that 100 mg/kg/day was the no observed adverse effect level (NOAEL). Despite the slight bodyweight effects (males and females) and dermal reactions at the application site (females) at this dose level, the general condition of parental animals, food consumption and organ weights showed no adverse effects of treatment, and there were no macroscopic or microscopic lesions that could be attributed to treatment with the test substance. In the absence of any impairment of reproductive function, the high dose of 100 mg/kg/day was considered to be the no observed effect level (NOEL) in terms of reproductive function and fertility.
Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Additional information

Screening for reproduction/developmental effects (OECD 421)

Patten (2010) performed a reproductive/developmental screening study via the dermal route of exposure according to OECD 421 guideline. In this GLP study male and female Crl:CD(SD) rats (10 animals/sex/dose) were exposed to 10 mg/kg/day, 50 mg/kg/day or 100 mg/kg/day. Control animals received concurrent vehicle (water). The rats were exposed on a daily basis (7 days per week) at 6 hours per day, except for females on Day 21 of mating and Day 2 and 3 of lactation when exposure was only three hours. No significant adverse effects were observed in parental animals. In offspring, the post-implantation survival rate was low which was attributed to two litters at 100 mg/kg/day. It was considered likely that the increased post-implantation loss in these two high dose litters is fortuitous and is exaggerated by the small sample size, and therefore considered unlikely to be related to treatment. However, the potential for higher in utero loss at 100 mg/kg/day cannot be resolved within the context of the design of this screening study. It was concluded that in this study, in terms of general toxicity, 100 mg/kg/day was the no observed adverse effect level (NOAEL). Despite the slight bodyweight effects (males and females) and dermal reactions at the application site (females) at this dose level, the general condition of parental animals, food consumption and organ weights showed no adverse effects of treatment, and there were no macroscopic or microscopic lesions that could be attributed to treatment with the test substance. In the absence of any impairment of reproductive function, the highest dose of 100 mg/kg/day was considered to be the no observed effect level (NOEL) in terms of reproductive function and fertility.

Extended one-generation reproductive toxicity study

A reproductive/developmental toxicity screening study (according to OECD guideline 421) has been performed via dermal route of exposure. No adverse effects were observed at the highest dose tested (dermal exposure of 100 mg/kg bw/day).  There is no developmental toxicity at the highest concentration tested of 100 mg/kg bw/day. A key prenatal development toxicity study was performed according to OECD guideline 414. Oral administration of the test substance to pregnant rats from gestation day 3 to day 19, at dose levels up to 200/125 mg/ kg bw/day was associated with premature death of two animals at 200 mg/kg bw/day. The NOEL for the pregnant female rats was considered to be at least 100 mg/kg bw/day. In utero survival of the dev eloping conceptus was unaffected by maternal treatment with 200/125 mg/kg bw/day. No changes in the measured fetal parameters or embryofetal development were detected up to 200/125 mg/kg bw/ day. The NOEL for development toxicity was therefore considered to be at least 125 mg/kg bw/day. T he substance is classified as mild skin irritant based on a key in vivo skin irritation study (according to OECD guideline 404) and showed moderate to severe irritation in the 90-day dermal repeated dose toxicity study at resp. 50 and 160 mg/kg bw/d dose levels. Therefore, testing at higher concentrations would not be justifiable. In addition, reproductive tissues (including gonads, uterus, epididymides, prostate, and if present, seminal vesicle) were examined for gross pathology and/or histopathology in both 90-day repeated dose toxicity studies and no adverse effects were noted. Moreover, exposure to the test substance is considered to be limited. Therefore, based on the above, the test substance is considered not to be tested in an extended one generation reproduction toxicity study.

Effects on developmental toxicity

Description of key information

One K1 prenatal development toxicity study was performed in rats according to the OECD 414 guideline.

The oral (gavage) administration of the test substance to pregnant rats from gestation Days 3 to 19, at dose levels of 10, 100 or 200/125 mg/kg bw/day was associated with the deaths of two animals at 200 mg/kg bw/day. The No Observed Effect Level (NOEL) for the pregnant female was therefore considered to be at least 100 mg/kg bw/day. In-utero survival of the developing conceptus was unaffected by maternal treatment with 200/125 mg/kg bw/day. No changes in the measured fetal parameters or embryofetal development were detected at 10, 100 or 200/125 mg/kg bw/day. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was therefore considered to be at least 125 mg/kg bw/day.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-06-21 to 2017-08-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
22 January 2001
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
August 1998
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 NohSan No 8147
Version / remarks:
24 November 2000
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No 440/2008 of 30 May 2008 test methods pursuant to Regulations (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 3717
- Expiration date of the lot/batch: 01 December 2019
- Purity: 100%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
- Solubility and stability of the test substance in the solvent/vehicle: For the purpose of the study the test item was prepared at the appropriate concentrations as a solution in distilled water. The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK Analytical Services (under Envigo study number: QJ79QM). Results showed the formulations to be stable for at least twenty one days when stored at 4 °C in the dark.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Bulk formulations were prepared twice and then divided into daily aliquots and stored at approximately 4 °C in the dark for the 10, 100 and 200 mg/kg bw/day formulations. As the dose level was reduced from 200 to 125 mg/kg bw/day so close to the end of the study the 125 mg/kg bw/day formulations were prepared daily for the remainder of the study (07 July 2017 to 11 July 2017).

OTHER SPECIFICS:
No correction for purity was made.
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD (SD) IGS BR strain
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: 96 time-mated female Sprague-Dawley Crl:CD (SD) IGS BR strain rats were obtained from Charles River (UK) Limited, Margate, Kent. Animals were delivered in two batches containing females prior to Day 3 of gestation.
- Age at study initiation: no data
- Weight at study initiation: 172 to 300 g on arrival
- Fasting period before study: no data
- Housing: individually in solid-floor polypropylene cages with stainless steel mesh lids furnished with softwood flakes (Datesand Ltd., Cheshire, UK). Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK)
- Diet (e.g. ad libitum): ad libitum, pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK)
- Water (e.g. ad libitum): ad libitum, mains drinking water from polycarbonate bottles attached to the cage
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 ºC
- Humidity (%): 50 ± 20%
- Air changes (per hr): at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): low intensity fluorescent lighting, 12 hours continuous light/12 hours darkness.

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Bulk formulations were prepared twice and then divided into daily aliquots and stored at approximately 4 °C in the dark for the 10, 100 and 200 mg/kg bw/day formulations. As the dose level was reduced from 200 to 125 mg/kg bw/day so close to the end of the study the 125 mg/kg bw/day formulations were prepared daily for the remainder of the study (07 July 2017 to 11 July 2017).

Treatment volume: 5 mL/kg

VEHICLE
- Concentration in vehicle: 2, 20, 40/25#
# dose level reduced from 200 mg/kg bw/day from Day 17
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item concentration in the test samples was determined by high performance liquid chromatography-mass spectrometry (HPLC-MS) using an external standard technique. The test item gave a chromatographic profile consisting of two peaks. Samples were taken of each bulk test item formulation and the first daily preparation formulation (125 mg/kg bw/day) and were analyzed for concentration of the test substance.

Preparation of Calibration Standards
Stock solutions of test item in dilution solvent were prepared for external standard calibration. An aliquot, approximately 0.1 g of test item was exactly weighed into a 100 mL volumetric flask and brought to volume with dilution solvent to yield a solution with a concentration of 1 mg/mL. Aliquots of this stock standard solution were used to prepare working standard solutions in dilution solvent with a concentration range of 0.05 mg/mL to 0.25 mg/mL.
On each occasion standard solutions derived from two stock standard solutions were used for calculation.
Calibration solutions were injected onto the instrument, at the beginning and end of each sample analysis sequence as a minimum, using the conditions detailed in the instrument parameters section.
To assess the calibration range of the method, a range of standard solutions were prepared in dilution solvent from a stock solution of 1.167 mg/mL by serial dilution covering the concentration range 0.05835 mg/mL to 0.3360 mg/mL.

Preparation of Test Samples
The formulations received were diluted with dilution solvent. An aliquot of test item formulation was accurately weighed into a volumetric flask and brought to volume with dilution solvent this was then shaken to dissolve. Where necessary, sample solutions were further diluted with dilution solvent to achieve the working concentration.

Preparation of Accuracy and Precision Samples
This was performed under study number QJ79QM.

Instrumentation Parameters
HPLC System : Agilent Technologies 1200 MSD, incorporating autosampler and workstation
Mass selective detector
Source: electrospray
Fragmentation energy: 70volts
Polarity: positive
Mode: single ion mode with 422.4 amu
Gas temperature: 350”C
Drying gas: 12.5 litre/minute
Nebuliser pressure: 40 psi
Capillary voltage: 2500 volts
Gain: 1
Column: Kinetix C8, 2.6 µ, (50 x 3 mm id)
Column temperature: 30”C
Gradient elution: eluent A: 0.1% formic acid in water
eluent B: acetonitrile
Time (minutes) % A) % B
0 100 0
5 20 80
10 100 0
Flow rate: 0.5 mL/min
Injection volume: 15.00 µL
Retention time: approximately 0.5 and 3.5 minutes

Data Evaluation and Calculations
The peak area response for the Test Item in each calibration standard chromatogram was measured. Calibration curves were constructed by non-linear regression of calibration standard response versus calibration standard concentration. The area response of the peak observed at the characteristic retention time for Test Item in sample and procedural recovery chromatograms was measured.

Concentration of Dose Formulations
For each analysis occasion, freshly prepared test formulations were analyzed. Duplicate samples were analyzed in accordance with the analytical procedure. Samples were disposed of once satisfactory results were achieved.

RESULTS
Concentration of Dose Formulations
The mean concentrations were within applied limits ±10%, confirming accurate formulation.

CONCLUSION
The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, linearity of detector response, method accuracy and precision during study number QJ79QM.
The homogeneity and stability was confirmed during study number QJ79QM.
The mean concentrations of Test Item in test formulations analyzed for the study were within ±10% of nominal concentrations, confirming accurate formulation.
Details on mating procedure:
Pre-mated animals were ordered. Animals were delivered in two batches containing females prior to Day 3 of gestation. The day that positive evidence of mating was observed was designated Day 0 of gestation.
Duration of treatment / exposure:
16 days, from Day 3 to Day 19 of gestation
Frequency of treatment:
daily
Duration of test:
16 days
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
control group, vehicle only
Dose / conc.:
10 mg/kg bw/day (nominal)
Remarks:
low dose group
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
intermediate dose group
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
high dose group, from start of treatment until day 17 of gestation
Dose / conc.:
125 mg/kg bw/day (nominal)
Remarks:
high dose group, from day 17 of gestation until end of treatment
No. of animals per sex per dose:
24 females/dose group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were chosen in collaboration with the Sponsor Representative and were based on available toxicity data including a Preliminary Oral (Gavage) Pre-Natal Development Toxicity Study in the Rat (Envigo Study Number KM11LR) and a 90 Day Oral (Gavage) Toxicity Study in the Rat (Envigo Study Number QJ79QM). The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.
- Rationale for animal assignment: The animals were randomly allocated to treatment groups using a randomization procedure based on stratified body weight to ensure similarity between the treatment groups.

Maternal examinations:
CLINICAL OBSERVATIONS
- Time schedule: once daily during the gestation period, immediately before and soon after dosing and one hour post dosing during the dosing period
- Parameters: overt signs of toxicity, ill-health or behavioral changes

BODY WEIGHT
- Time schedule: on Day 3 (before the start of treatment) and on Days 4, 5, 8, 11, 14 and 17 of gestation. Body weights were also recorded for surviving animals at terminal kill (Day 20).

FOOD CONSUMPTION
- Time schedule: at Day 3, 5, 8, 11, 14, 17 and 20 of gestation.

WATER CONSUMPTION
- Time schedule: daily by visual inspection of the water bottles for any overt changes.

NECROPSY
- All surviving animals were killed by carbon dioxide asphyxiation followed by cervical dislocation on Day 20 of gestation.
- All animals were subjected to a full external and internal examination (including examination of the uterine contents) and any macroscopic abnormalities were recorded
Ovaries and uterine content:
- The ovaries and uteri of pregnant females were removed, examined and the following data recorded: Number of corpora lutea; Number, position and type of intrauterine implantation; Fetal sex; External fetal appearance; Fetal weight; Placental weight; Gravid uterus weight
- Implantation types were divided into:
Early Death: No visible distinction between placental/decidual tissue and embryonic tissue
Late Death: Separate embryonic/fetal and placental tissue visible
Dead Fetus: A fetus that had died shortly before necropsy. These were included as late deaths for reporting purposes

All implantations and viable fetuses were numbered according to their intrauterine position.
Fetal examinations:
- The fetuses were killed by subcutaneous injection of sodium pentobarbitone.
- Fetuses from each litter were divided into two groups and examined for skeletal alterations and soft tissue alterations.
- Alternate fetuses were identified using an indelible marker and placed in Bouin’s fixative. Fetuses were subsequently transferred to distilled water and examined for visceral anomalies under a low power binocular microscope and then stored in 10% Buffered Formalin.
- The remaining fetuses were identified using cardboard tags marked with chinagraph pencil and placed into 70% IMS in distilled water. The fetuses were subsequently eviscerated, processed and the skeletons stained with alizarin red S before being transferred to 50% glycerol for examination of skeletal development and anomalies and storage.
Statistics:
The following parameters were analyzed statistically, where appropriate, using the test methods outlined below:
Female body weight change, food consumption and gravid uterus weight: Shapiro Wilk normality test and Bartlett’s test for homogeneity of variance and one way analysis of variance, followed by Dunnett’s multiple comparison test or, if unequal variances were observed, on alternative multiple comparison test.
All caesarean necropsy parameters and fetal parameters: Kruskal-Wallis non-parametric analysis of variance; and a subsequent pairwise analysis of control values against treated values using the Mann-Whitney ‘U’ test, where significance was seen.
Fetal evaluation parameters, including skeletal or visceral findings: Kruskal-Wallis non-parametric analysis of variance and Mann-Whitney ‘U’ test.
Probability values (p) are presented as follows:
p<0.01 **
p<0.05 *
p≥0.05 (not significant)
Indices:
Pre and Post Implantation Loss:
Percentage pre-implantation loss was calculated as: [(number of corpora lutea - number of implantations)/number of corpora lutea] x 100

Percentage post-implantation loss was calculated as: ([number of implantations - number of live fetuses)/number of implantations] x 100

Sex ratio: % male fetuses (sex ratio) = (Number of male fetuses/Total number of fetuses) x 100
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs were apparent in any animal during the course of the study.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
Two animals treated with 200 mg/kg bw/day were found dead on the 07 July 2017 (Days 15 and 14 of gestation respectively). There were no clinical signs noted in either of these animals prior to death.
There were no further unscheduled deaths
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No adverse effect on body weight development was evident in animals from any treatment group.
Animals treated with 10 mg/kg bw/day showed a statistically significant increase (p<0.01) in cumulative body weight gain from Days 3 to 11 of gestation. An increase in body weight gain is considered not to reflect an adverse effect of treatment and was therefore considered not to be of toxicological significance.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No adverse effect on food consumption was evident in animals from any treatment group.
Animals treated with 200 mg/kg bw/day exhibited a statistically significant reduction (p<0.01) in food consumption from Days 3 to 5 of gestation. As recovery was evident thereafter and all food consumption data generated for this treatment group was within the historical control data range this reduction was considered not to be of any toxicological significance.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Daily visual inspection of water bottles did not reveal any overt intergroup differences.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The two animals which were found dead on Days 15 and 14 of gestation exhibited fluid contents in the stomach and patchy pallor on the liver.
With the exception of one instance of yellow colored contents in the stomach in one animal treated with 200/125 mg/kg bw/day, no macroscopic abnormalities were detected in any surviving animal at necropsy.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There was no obvious effect of maternal treatment on litter data as assessed by pre- and post-implantation losses at 10, 100 or 200/125 mg/kg bw/day.
Total litter losses by resorption:
not specified
Early or late resorptions:
no effects observed
Description (incidence and severity):
There was no obvious effect of maternal treatment on litter data as assessed by in utero offspring survival (as assessed by the mean numbers of early or late resorptions) at 10, 100 or 200/125 mg/kg bw/day.
Dead fetuses:
not specified
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
not specified
Other effects:
no effects observed
Description (incidence and severity):
There was no obvious effect of maternal treatment on litter data as assessed by numbers of implantations, in-utero offspring survival (as assessed by the mean numbers of early or late resorptions), live litter size, sex ratio and pre and post-implantation losses at 10, 100 or 200/125 mg/kg bw/day.
Intergroup differences for mean fetal, litter or placental weights did not indicate any obvious effects of maternal treatment at 10, 100 or 200/125 mg/kg bw/day
Key result
Dose descriptor:
NOEL
Effect level:
>= 100 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
mortality
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Intergroup differences for mean fetal did not indicate any obvious effects of maternal treatment at 10, 100 or 200/125 mg/kg bw/day.
Reduction in number of live offspring:
not examined
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There was no obvious effect of maternal treatment on litter data as assessed by sex ratio at 10, 100 or 200/125 mg/kg bw/day.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There was no obvious effect of maternal treatment on litter data as assessed by live litter size at 10, 100 or 200/125 mg/kg bw/day.
Intergroup differences for mean fetal, litter or placental weights did not indicate any obvious effects of maternal treatment at 10, 100 or 200/125 mg/kg bw/day.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
Neither the type, incidence nor distribution of external finding apparent for fetuses at Day 20 of gestation indicated an effect of maternal treatment on fetal development at 10, 100 or 200/125 mg/kg bw/day.
Skeletal malformations:
no effects observed
Description (incidence and severity):
Skeletal examinations of fetuses on Day 20 of gestation did not indicate any obvious effect of maternal treatment on fetal development at 10, 100 or 200/125 mg/kg bw/day.
Statistically significant reductions (p<0.05-0.01) in the number of fetuses exhibiting incomplete ossification of the hyoid bone of the skull, zygomatic process of squamosal of the skull and metacarpals were noted in animals treated with 10, 100 and 200/125 mg/kg bw/day, however, these were not regarded as evidence of developmental toxicity as these decreases were closer to the expected range for these parameters.
Visceral malformations:
no effects observed
Description (incidence and severity):
Visceral examinations of fetuses on Day 20 of gestation did not indicate any obvious effect of maternal treatment on fetal development at 10, 100 or 200/125 mg/kg bw/day.
Other effects:
not specified
Key result
Dose descriptor:
NOEL
Effect level:
>= 125 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
external malformations
skeletal malformations
visceral malformations
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Group Mean Litter Data Values

Dose Level (mg/kg bw/day)

 

Number of Corpora Lutea

Number of Implants

Number of Embryonic/Fetal Deaths

Implantation Loss
%

Number of Live Implants

%
Male
Fetuses

Mean Male Fetal Weight (g)

Mean Female Fetal Weight (g)

Mean Fetal Weight (g)

MeanPlacentalWeight
(g)

Litter Weight (g)

TotalPlacentalWeight
(g)

Early

Late

Total

Pre

Post

Male

Female

Total

0 (Control)

mean

16.8

13.4

0.2

0.0

0.3

19.9

1.9

6.9

6.2

13.1

52.7

4.115

3.882

4.012

0.582

52.594

7.590

sd

2.3

1.6

0.5

0.2

0.5

9.4

4.0

2.1

2.1

1.7

14.3

0.204

0.251

0.201

0.072

6.751

0.952

n

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

23#

10

mean

17.2

13.8

0.1

0.1

0.3

19.6

1.8

6.8

6.7

13.5

50.9

4.135

3.959

4.051

0.589

54.477

7.916

sd

2.5

2.3

0.4

0.4

0.6

10.6

4.6

2.6

2.6

2.3

16.5

0.276

0.282

0.248

0.066

8.989

1.502

n

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

100

mean

17.7

13.6

0.1

0.0

0.2

22.1

1.2

6.7

6.7

13.4

50.0

4.151

3.960

4.052

0.578

54.284

7.700

sd

2.7

1.5

0.3

0.2

0.4

11.5

2.7

2.0

1.9

1.4

14.1

0.247

0.234

0.224

0.090

5.662

1.121

n

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

200/125

mean

16.5

13.8

0.2

0.1

0.3

16.0

2.4

6.9

6.6

13.5

52.0

4.204

3.985

4.103

0.614

55.299

8.270#

sd

2.7

2.7

0.7

0.3

0.7

9.7

5.5

1.9

2.7

2.8

13.9

0.327

0.296

0.307

0.085

11.918

1.911

n

22

22

22

22

22

22

22

22

22

22

22

22

22

22

22

22

21

#= Total placental weight for Females 22 and 77 excluded from group mean as one placenta not weighed in error

Conclusions:
The oral (gavage) administration of the test substance to pregnant rats from gestation Days 3 to 19, at dose levels of 10, 100 or 200/125 mg/kg bw/day was associated with the deaths of two animals at 200 mg/kg bw/day.
The No Observed Effect Level (NOEL) for the pregnant female was therefore considered to be at least 100 mg/kg bw/day. In-utero survival of the developing conceptus was unaffected by maternal treatment with 200/125 mg/kg bw/day. No changes in the measured fetal parameters or embryofetal development were detected at 10, 100 or 200/125 mg/kg bw/day.
The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was therefore considered to be at least 125 mg/kg bw/day.
Executive summary:

The study was performed to investigate the effects of the test item on embryonic and fetal development following repeated administration by gavage to the pregnant female during gestation including the period of organogenesis.

The study was designed to comply with the following guidelines:

·        US EPA Health Effects Test Guideline OPPTS 870.3700, ‘Prenatal Developmental Toxicity Study’ (August 1998)

·        Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 NohSan No 8147, (24 November 2000)

·        OECD Guidelines for Testing of Chemicals, No 414, ‘Prenatal Developmental Toxicity Study’ (adopted 22 January 2001)

·        Commission Regulation (EC) No 440/2008 of 30 May 2008 test methods pursuant to Regulations (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)

Methods

The test item was administered by gavage to three groups each of twenty-four time mated Sprague-Dawley Crl:CD®(SD) IGS BR strain rats, between Days 3 and 19 of gestation inclusive at dose levels 10, 100, and 200 mg/kg bw/day (reduced to 125 mg/kg bw/day from Day 17 (animals 73-80), Day 16 (animals 81-88) or Day 15 (animals 89-96) of gestation onwards due to unscheduled mortality). Similar effects were also noted on a Ninety Day Repeated Dose study, Envigo Study Number: QJ79QM where dose levels were reduced from 200 to 150 mg/kg bw/day from Day 31 (males) and Day 30 (females), dose levels were subsequently reduced on this study again to 75 mg/kg bw/day from Day 36 (males) and Day 35 (females). A further group of twenty-four time mated females was exposed to the vehicle only (distilled water) over the same treatment period to serve as a control.

Clinical signs, body weight change, food and water consumptions were monitored during the study. 

All surviving females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weights, fetal weight, sex and external and internal macroscopic appearance were recorded. Half of each litter were examined for detailed skeletal development and the remaining half were subjected to detailed visceral examination.

 

Results…….

Mortality

Two animals treated with 200 mg/kg bw/day were found dead on the 07 July 2017 (Days 15 and 14 of gestation respectively). There were no clinical signs noted in either of these animals prior to death.

There were no further unscheduled deaths.

Clinical Observations

No clinical signs were apparent in any animal during the course of the study.

Body Weight

No adverse effect on body weight development was evident in animals from any treatment group.

Food Consumption

No adverse effect on food consumption was evident in animals from any treatment group.

Water Consumption

Daily visual inspection of water bottles did not reveal any overt intergroup differences.

Post Mortem Studies

The two animals which were found dead on Days 15 and 14 of gestation exhibited fluid contents in the stomach and patchy pallor on the liver.

With the exception of one instance of yellow colored contents in the stomach in one animal treated with 200/125 mg/kg bw/day, no macroscopic abnormalities were detected in any surviving animal at necropsy.

Litter Data and Litter Placental and Fetal Weights

There was no obvious effect of maternal treatment on litter data as assessed by numbers of implantations,in-uterooffspring survival (as assessed by the mean numbers of early or late resorptions), live litter size, sex ratio and pre and post-implantation losses at 10, 100 or 200/125 mg/kg bw/day.

Intergroup differences for mean fetal, litter or placental weights did not indicate any obvious effects of maternal treatment at 10, 100 or 200/125 mg/kg bw/day.

Fetal Examination

External Findings

Neither the type, incidence nor distribution of external finding apparent for fetuses at Day 20 of gestation indicated an effect of maternal treatment on fetal development at 10, 100 or 200/125 mg/kg bw/day.

Detailed Visceral Examinations

Visceral examinations of fetuses on Day 20 of gestation did not indicate any obvious effect of maternal treatment on fetal development at 10, 100 or 200/125 mg/kg bw/day.

Detailed Skeletal Examination

Skeletal examinations of fetuses on Day 20 of gestation did not indicate any obvious effect of maternal treatment on fetal development at 10, 100 or 200/125 mg/kg bw/day.

Conclusion

The oral (gavage) administration of JEFFAMINE T-403 to pregnant rats from gestation Days 3 to 19, at dose levels of 10, 100 or 200/125 mg/kg bw/day was associated with the deaths of two animals at 200 mg/kg bw/day. The No Observed Effect Level (NOEL) for the pregnant female was therefore considered to be at least 100 mg/kg bw/day.

In-uterosurvival of the developing conceptus was unaffected by maternal treatment with 200/125 mg/kg bw/day. No changes in the measured fetal parameters or embryofetal development were detected at 10, 100 or 200/125 mg/kg bw/day. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was therefore considered to be at least 125 mg/kg bw/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
125 mg/kg bw/day
Study duration:
subacute
Species:
rat
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

A reproductive / developmental toxicity screening study (according to OECD 421) has been performed. No adverse effects were observed at the highest dose tested (dermal exposure of 100 mg/kg bw/d). Results from the OECD 421 screening assessment study conclude that there is no developmental toxicity at the highest concentration tested of 100 mg/kg/day.

The substance is classified as a mild skin irritant (CLP cat 3) and showed moderate to severe irritation in the 90 d dermal repeated dose test at resp. 50 and 160 mg/kg bw/d dose levels. Therefore, testing at higher concentrations is not justifiable. In addition, reproductive tissues (including gonads, uterus, epididymides, prostate, and if present, seminal vesicles) were examined for gross pathology in the 90 day repeated dose study and no adverse effects were noted.

The registrant considers that this endpoint has been adequately tested through the use of the screening study and that no additional studies are required. Moreover, exposure to this substance is considered to be limited.

Prenatal development toxicity study

The test item was administered by oral gavage to three groups each of twenty-four time mated Sprague-Dawley Crl:CD®(SD) IGS BR strain rats, between Days 3 and 19 of gestation inclusive at dose levels 10, 100, and 200 mg/kg bw/day (reduced to 125 mg/kg bw/day from Day 17 (animals 73-80), Day 16 (animals 81 -88) or Day 15 (animals 89 -96) of gestation onwards due to unscheduled mortality) (according to OECD guideline 414, K1, Envigo, 2018). Similar effects were also noted on a Ninety Day Repeated Dose study, Envigo Study Number: QJ79QM where dose levels were reduced from 200 to 150 mg/kg bw/day from Day 31 (males) and Day 30 (females), dose levels were subsequently reduced on this study again to 75 mg/kg bw/day from Day 36 (males) and Day 35 (females). A further group of twenty-four time mated females was exposed to the vehicle only (distilled water) over the same treatment period to serve as a control. Clinical signs, body weight change, food and water consumptions were monitored during the study. All surviving females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weights, fetal weight, sex and external and internal macroscopic appearance were recorded. Half of each litter were examined for detailed skeletal development and the remaining half were subjected to detailed visceral examination.

Two animals treated with 200 mg/kg bw/day were found dead on Days 15 and 14 of gestation respectively. There were no clinical signs noted in either of these animals prior to death. There were no further unscheduled deaths. No clinical signs were apparent in any animal during the course of the study. No adverse effect on body weight development was evident in animals from any treatment group. No adverse effect on food consumption was evident in animals from any treatment group. Daily visual inspection of water bottles did not reveal any overt intergroup differences. The two animals which were found dead on Days 15 and 14 of gestation exhibited fluid contents in the stomach and patchy pallor on the liver. With the exception of one instance of yellow colored contents in the stomach in one animal treated with 200/125 mg/kg bw/day, no macroscopic abnormalities were detected in any surviving animal at necropsy.

There was no obvious effect of maternal treatment on litter data as assessed by numbers of implantations, in-utero offspring survival (as assessed by the mean numbers of early or late resorptions), live litter size, sex ratio and pre and post-implantation losses at 10, 100 or 200/125 mg/kg bw/day. Intergroup differences for mean fetal, litter or placental weights did not indicate any obvious effects of maternal treatment at 10, 100 or 200/125 mg/kg bw/day.

Neither the type, incidence nor distribution of external finding apparent for fetuses at Day 20 of gestation indicated an effect of maternal treatment on fetal development at 10, 100 or 200/125 mg/kg bw/day. Visceral and skeletal examinations of fetuses on Day 20 of gestation did not indicate any obvious effect of maternal treatment on fetal development at 10, 100 or 200/125 mg/kg bw/day.

The oral (gavage) administration of the test substance to pregnant rats from gestation Days 3 to 19, at dose levels of 10, 100 or 200/125 mg/kg bw/day was associated with the deaths of two animals at 200 mg/kg bw/day. The No Observed Effect Level (NOEL) for the pregnant female was therefore considered to be at least 100 mg/kg bw/day. In-utero survival of the developing conceptus was unaffected by maternal treatment with 200/125 mg/kg bw/day. No changes in the measured fetal parameters or embryofetal development were detected at 10, 100 or 200/125 mg/kg bw/day. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was therefore considered to be at least 125 mg/kg bw/day.

Toxicity to reproduction: other studies

Description of key information

No other studies are available.

Justification for classification or non-classification

Based on the available data and according to the criteria of the CLP Regulation, the test substance should not be classified for toxicity to reproduction nor for developmental toxicity.