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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
For the test substance no standard OECD TG 416 two generation study on reproduction toxicology is available. In accordance with section 1 of Annex XI this study is not scientifically justified because several experimental data on reproductive toxicity are available, including a one-generation study (OECD 415) and several developmental toxicity studies. Reproduction toxicity is only seen at maternally toxic doses and no irreversible effects are observed. It is not expected that the NOAEL for reproductive toxicity in a two generation study will be lower than in the present studies or that classification for reproductive toxicity will be warranted, because all parameters have been extensively studied.

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of 3-(4-hydroxy-4-methylpentyl)cyclohex-3-ene-1-carbaldehyde and 4-(4-hydroxy-4-methylpentyl)cyclohex-3-enecarbaldehyde
EC Number:
915-617-9
Molecular formula:
C13H22O2
IUPAC Name:
Reaction mass of 3-(4-hydroxy-4-methylpentyl)cyclohex-3-ene-1-carbaldehyde and 4-(4-hydroxy-4-methylpentyl)cyclohex-3-enecarbaldehyde
Test material form:
liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, UK
- Age at study initiation: (P) males ca. 6 weeks, females ca. 10 weeks
- Weight at study initiation: (P) Males: 191 - 255 g; Females: 211 - 271 g
- Housing: initially, in groups of 4 in polypropylene cages with stainless steel grid floors and tops. During the mating, animals were housed in similar cages on 1:1 male:female basis. After mating males were transferred to the original cages; females were housed individually during gestation and lactation in polypropylene cages with solid floors and stainless steel lids.
- Diet: pelleted diet (Rodent PMI 5002 (certified) diet, ad libitum
- Water: Mains drinking water, ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS
- Formulations were prepared weekly.

VEHICLE
- Concentration in vehicle: 0, 6.25, 25 and 125 mg/mL
- Amount of vehicle: 4 mL/kg bw
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: a maximum of 21 days
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 1 of pregnancy
- After successful mating each pregnant female was caged individually during gestation and lactation in polypropylene cages with solid floors and stainless steel lids, furnished with softwood flakes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of the test substance in arachis oil was determined by gas chromatography using an external standard technique.
Duration of treatment / exposure:
76 days pre-mating, maximal 21 days mating, males were killed and examined upon evidence of successful mating, females and offspring were killed and examined on day 21 post-partum. Non-pregnant females were killed and examined after day 25 post-coitum.
Frequency of treatment:
Daily (except for females during littering/parturition)
Details on study schedule:
Age at mating of the mated animals in the study: mating was performed on day 76 of treatment.
Doses / concentrationsopen allclose all
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on the results of a 14-day dose-range finding study
- Rationale for animal assignment: the animals were allocated to dose groups using a randomisation procedure based on stratified body weights.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: the animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before and after the dosing, and 1 and 5 hours post-dosing during the working week. Animals were observed immediately before and after dosing and 1 hour post-dosing at the weekends or public holidays.

BODY WEIGHT: Yes
- Time schedule for examinations: day 0, then weekly for males until termination. Females were weighed weekly during maturation and daily during mating. Once mating was evident, body weights were recorded on days 1, 4, 7, 14 and 21 of post-coitum and post-partum.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: During the maturation period, weekly food consumption was recorded for each cage. For females showing evidence of mating, food consumption was recorded for the period covering days 1 - 7, 7 - 14 and 14 - 21 post-coitum. For females with live litters, food consumption was recorded for the period covering days 1 - 7, 7 - 14 and 14 - 21 post-partum.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water intake was observed daily by visual inspection of water bottles for any overt change.

Oestrous cyclicity (parental animals):
During mating, a vaginal smear was prepared for each female daily and the stage of the oestrous cycle was recorded.
Sperm parameters (parental animals):
Parameters examined in P male parental generations: testis weight, epididymis weight, numbers of homogenisation resistant spermatids, sperm motility, sperm morphology.
Litter observations:
PARAMETERS EXAMINED
- The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, the detachment and unfolding of pinna, incisor eruption and eyelid separation, reflexological response to stimuli by assessing surface righting reflex on Day 1 post-partum and air righting reflex on Day 17 post-partum.
- Pupillary reflex and auditory startle response were performed on day 21 post-partum.

GROSS EXAMINATION OF DEAD PUPS
- For external and internal abnormalities.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals; after successful mating.
- Maternal animals: All surviving animals; on day 21 post-partum; for non-pregnant females on or after day 25 post-coitum.

GROSS NECROPSY
- Gross necropsy consisted of full external and internal examinations.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were prepared for microscopic examination and weighed, respectively: cervix, coagulating gland, epididymides, ovaries, pituitary gland, prostate, seminal vesicles, testes, uterus, vagina.
Postmortem examinations (offspring):
SACRIFICE
The off-spring was sacrificed on day 21 after birth.

GROSS NECROPSY
Gross necropsy consisted of full external and internal examinations.
Statistics:
Linear regression analysis, followed by ANOVA incorporating Levene's test for homogeneity of variance was used for data on organ weights, weekly body weight, litter weights, offspring body weights. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett's test. Where Levene's test showed unequal variances, the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney "U" test.
The non-parametric methods were also used to analyse implantation loss, offspring sex ratio, litter size and landmark developmental markers.
Chi-squared analysis was used for differences in the incidence of lesions occurring with an overall frequency of 1 or greater.
Kruskal-Wallis one-way non-parametric analysis of variance was used for the comparison of severity grades for the more frequently observed graded conditions.
Reproductive indices:
The following indices were calculated:
Mating index = (number of animals mated/number of animals paired) x 100 %
Pregnancy index = (number of pregnant females/number of animals mated) x 10 0%
Parturition index = (number of females delivering live offspring/number of pregnant females) x 100 %
Offspring viability indices:
The following indices were calculated:
Live birth index = (number of offspring alive on day 1/number of offspring born) x 100 %
Viability index 1 = (number of offspring alive on day 4/number of offspring alive on day 1) x 100 %
Viability index 2 = (number of offspring alive on day 7/number of offspring alive on day 4) x 100 %
Viability index 3 = (number of offspring alive on day 14/number of offspring alive on day 7) x 100 %
Viability index 4 = (number of offspring alive on day 21/number of offspring alive on day 14) x 100 %
Viability index 5 = (number of offspring alive on day 21/number of offspring alive on day 1) x 100 %

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Episodes of hunched posture, pilo-erection and tiptoe gait were evident in 500 mg/kg bw/day females during the final week of gestation.
Mortality:
mortality observed, treatment-related
Description (incidence):
One male treated with 500 mg/kg bw/day was killed in extremis on day 93. One female from this treatment group was found dead on day 97 and a further two females were killed in extremis on days 99 and 100 following difficulties during parturition
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gain for males of 500 mg/kg bw/day group was generally lower (average -22 %, range -9 % to -43 %) than control animals throughout much of the treatment period, with statistical differences observed in Weeks 4, 5, 6 and 10.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Females treated with 500 mg/kg bw/day showed a notable reduction in food consumption (average -21 %, range -17 % to -28 %) throughout lactation, with statistically significant differences throughout 3 weeks
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Parental animals: no treatment-related microscopic changes were observed in the parental animals.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on female oestrous cycles or on the type or proportion of females with anomalous oestrous cycle.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
There were no toxicologically significant effects on the concentration, motility or morphology of samples of epididymal sperm. There were no treatment-related effects on the concentration of homogenisation resistant epididymal or testicular spermatid count.
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating performance was good in all groups with the majority of animals mating within the first four days of pairing. Subsequent pregnancy rate was unaffected by treatment with only 1, 1, 1 and 2 females failing to achieve pregnancy in the control, 25, 100 and 500 mg/kg bw/day groups, respectively.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
> 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
No adverse effects on fertility were noted at the highest tested dose.
Key result
Dose descriptor:
NOAEL
Remarks:
repeated dose toxicity
Effect level:
25 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Repeated dose toxicity was seen at 100 mg/kg bw based on gestation length increase.

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Skin sloughing was detected in offspring during the first week of lactation in all treatment groups (more pronounced in the high dose group) together with multiple ridges along the tail in 500 and 100 mg/kg bw/day litters. Swollen ears became apparent in 500 and 100 mg/kg bw/day litters together with the premature opening of eyes and sparse fur coverage in 500 mg/kg bw/day litters.
A delay in the onset (+ 2.1 days) and completion ( + 2.8 days) of pinna unfolding was evident in 500 mg/kg bw/day offspring together with a reduction in the number of offspring passing surface righting, air righting and pupil reflex. A total of eight 500 mg/kg bw/day litters had not fully completed eye opening by weaning.
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Life birth index for 500 mg/kg bw/day females was significantly lower (-15 %) than control animals with litter size continuing to be statistically lower than control animals throughout lactation. Of the high dose females that gave birth to live litters, six females had a total litter loss, predominantly between birth and day 1.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gain at 500 mg/kg bw/day was lower (-58%) than control animals for the first week of age and again from day 14 to weaning (day 21 of age) (-17%). Litter weight, at this treatment group, was notably lower than control animals throughout lactation.
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No treatment-related changes were detected.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Offspring from females treated with 500 and 100 mg/kg bw/day showed skin sloughing at necropsy. Offspring from females treated with 500 mg/kg bw/day also showed sparse fur coverage.
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
Acanthosis and hyperkeratosis were seen in relation to treatment for the skin of male and female F1 generation animals treated with 500 and 100 mg/kg bw/day.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Remarks:
Developmental
Generation:
F1
Effect level:
25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
In the one-generation study with rats, the NOAEL for parental toxicity was set at 25 mg/kg bw/day, based on gestation length increase in the 100 and 500 mg/kg bw dose groups. There was no advserse effect on fertility, therefore the NOAEL for fertility is set at the higest dose level, 500 mg/kg bw/day. The NOAEL for developmental effects was set at 25 mg/kg bw/day, based on the skin effects (skin peeling and flaking, and acanthosis and hyperkeratosis observed at necropsy) in male and female F1 animals at 100 mg/kg bw.
Executive summary:

Reproductive toxicity of the test substance was studied in a study with Sprague-Dawley rats, conducted according to OECD Guideline 415 in compliance with GLP. The substance was administered at dose levels of 0, 25, 100 and 500 mg/kg bw/day as a solution in arachis oil to groups of 24 rats/sex/dose for 76 days pre-mating and during maximum 21 days mating, after which males were killed, while females and subsequent offspring were killed and examined on day 21 post-partum. Non-pregnant females were killed and examined after day 25 post-coitum. One male treated with 500 mg/kg bw/day was killed in extremis on day 93. One female from this treatment group was found dead on day 97 and a further two females were killed in extremis on days 99 and 100 following difficulties during parturition. Episodes of hunched posture, pilo-erection and tiptoe gait were evident in 500 mg/kg bw/day females during the final week of gestation. Body weight gain for males of the 500 mg/kg bw/day group was generally lower (average -22 %, range -9 % to -43 %) than control animals throughout much of the treatment period, with statistical differences observed in weeks 4, 5, 6 and 10. Females treated with 500 mg/kg bw/day showed a notable reduction in food consumption (average -21 %, range -17 % to -28 %) throughout lactation, with statistically significant differences throughout 3 weeks. There were no treatment-related effects on female estrous cycles or on the type or proportion of females with an anomalous estrous cycle, or toxicologically significant effects on the concentration, motility or morphology of samples of epididymal sperm. There were no treatment-related effects on the concentration of homogenisation resistant epididymal or testicular spermatid counts. Mating performance was good in all groups with the majority of animals mating within the first four days of pairing. Subsequent pregnancy rate was unaffected by treatment with only 1, 1, 1 and 2 females failing to achieve pregnancy in the control, 25, 100 and 500 mg/kg bw/day groups, respectively. An increased gestation length was observed in the 100 and 500 mg/kg bw dose groups. At necropsy, the adult male treated with 500 mg/kg bw/day that was killed in extremis showed gaseous distension in the gasto-intestinal tract. The female treated with 500 mg/kg bw/day that was found dead around parturition had 21 foetuses in-utero. The two females from this treatment group that were killed in extremis both had dead/inactive fetuses in-utero and red/brown staining around the ano-genital region and dark contents in the stomach or enlarged adrenals and an absent rougae on the non-glandular region of the stomach. No treatment-related microscopic changes were observed in the parental animals. Based on the observed increased gestation length, the NOAEL for parental toxicity was set at 25 mg/kg bw/day.

Life birth index for 500 mg/kg bw/day females was significantly lower (-15 %) than control animals with litter size continuing to be statistically lower than control animals throughout lactation. Of the high dose females that gave birth to live litters, six females had a total litter loss, predominantly between birth and day 1. Skin sloughing was detected in offspring during the first week of lactation in all treatment groups (more pronounced in the high dose group) together with multiple ridges along the tail in 500 and 100 mg/kg bw/day litters. Swollen ears became apparent in 500 and 100 mg/kg bw/day litters together with the premature opening of eyes and sparse fur coverage in 500 mg/kg bw/day litters. A delay in the onset (+ 2.1 days) and completion (+ 2.8 days) of pinna unfolding was evident in 500 mg/kg bw/day offspring together with a reduction in the number of offspring passing surface righting, air righting and pupil reflex. A total of eight 500 mg/kg bw/day litters had not fully completed eye opening by weaning. Body weight gain in offspring at 500 mg/kg bw/day was lower (-58 %) than in control animals for the first week of age and again from day 14 to weaning (day 21 of age) (-17%). Litter weight, at this treatment group, was notably lower than control animals throughout lactation. Offspring from females treated with 500 and 100 mg/kg bw/day showed skin sloughing at necropsy. Offspring from females treated with 500 mg/kg bw/day also showed sparse fur coverage. Acanthosis and hyperkeratosis were seen in relation to treatment for the skin of male and female F1 generation animals treated with 500 and 100 mg/kg bw/day. Based on the skin effects in F1 offspring, the NOAEL was set at 25 mg/kg bw/day for the F1 generation. There was no advserse effect on fertility, therefore the NOAEL for fertility is set at the higest dose level, 500 mg/kg bw/day.