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Key value for chemical safety assessment

Effects on fertility

Description of key information

The toxicity of the test item Macrolex Rot E2G (CAS-No. 6829-22-7) on reproduction and/or development of the male and female rat was investigated in a reproduction/developmental toxicity screening test according to OECD guideline 421.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Principles of method if other than guideline:
The toxicity of the test item Macrolex Rot E2G (CAS-Nr. 6829-22-7) on reproduction and/or development of the male and female rat was investigated in a reproduction/developmental toxicity screening test according to OECD guideline 421.
GLP compliance:
yes
Specific details on test material used for the study:
Purity: 99.9%
Species:
rat
Strain:
other: Wistar rats: Crl: W1 (Han)
Details on species / strain selection:
One of the rodent species acceptable to the regulatory agencies. Historical control data for the strain are available at the Test Facility.
Sex:
male/female
Details on test animals and environmental conditions:
Test system
Species/strain: Wistar rats: Crl: WI (Han).
Supplier: Charles River Laboratories France, Domaine des Oncins, 69210 Saint-Germain-Nuelles, France.
Number of animals in the study: 80 (40 males and 40 females).
The virgin males were 11 weeks old at the start of treatment (body weight range 319 to 354 g).
The virgin females were 9 weeks old at the start of treatment (body weight range: 179 to 218 g).
Justification: One of the rodent species acceptable to the regulatory agencies. Historical control data for the strain are available at the Test Facility.

Animal husbandry
Housing: One air-conditioned room in a barrier protected unit (building K2).
Temperature: 22 + 3 °C (target range).
Relative humidity: 35 to 70 % (target range).
Air changes: At least 10 air changes per hour.
Lighting cycle: 12 hours light (artificial)/12 hours dark (except when required for technical acts).

Environmental conditions were within the targets throughout the study.
Caging: All animals were housed in plastic cages meeting European directive 2010/63/EU requirements as follows:
- Males and females: in groups of 5 of the same dose group before mating then singly during cohabitation (one male and one female per cage).
- Females: singly during gestation and lactation (one female with its litter). Tissue paper was added to the bedding towards the end of gestation.
Bedding: Dust-free sawdust (SDS/Dietex, Argenteuil, France) made from spruce tree wood, analysed at least twice a year for chemical and bacterial contaminants. Shredded paper (SDS/Dietex) was provided as enrichment. From day 20 of gestation, cellulose bedding (SERLAB, ALPHA-dri), analysed for chemical and bacterial contaminants was used.
Diet: Rat pelleted complete diet ad libitum (Diet reference A04C-10) sterilised by irradiation and analysed for a predefined list of chemical and bacteriological contaminants. Each batch of diet is supplied with a certificate of analysis which is verified and authorized for release by a veterinarian.
Water: Softened and filtered (0.2 μm) mains drinking water was available ad libitum (via an automatic watering system or bottles). Water is analysed twice a year for chemical and bacterial contaminants by Laboratoire Santé Environnement Hygiène de Lyon, France.
Contaminants: No known contaminants were present in the bedding, diet or water at levels which might have interfered with achieving the objectives of the study.
Route of administration:
oral: gavage
Vehicle:
other: % (w/v) Carboxymethylcellulose, 400-800 centipoises
Details on mating procedure:
Animals were paired on the basis of one male and one female from the same group for a maximum of 14 days.
Vaginal smears were taken daily from the females during cohabitation.
The day of mating was confirmed by the presence of sperm in a vaginal smear and was recorded and taken as day 0 of gestation (G0). Mated females were separated from the males once mating had been confirmed and smearing ceased.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Vehicle
Denomination: 1 % (w/v) Carboxymethylcellulose 400-800 centipoises.
Supplier: Sigma-Aldrich.
Storage: At room temperature (between +15 and +25 °C).
Water for injection:
Supplier: Laboratoire Aguettant
Storage: At room temperature (between +15 and +25 °C).
Frequency of preparation: At least once every 8 days.
Storage: At room temperature (between +15 and +25 °C).

Test item preparation
Preparation: The test item was prepared as a suspension in the vehicle at concentrations of 10, 30 and 100 mg/mL.
Correction factor for active ingredient: None.
Homogeneity of the test item in the vehicle: Suspensions at concentrations of 1 to 100 mg/mL have been shown to be homogeneous, according to AB20658 study.
Stability of the test item in the vehicle: 24 hours and 8 days at room temperature (+15 to +25 °C), protected from light or refrigerated (+2 and +8 °C) according to AB20658 study.
Frequency of preparation: At least once every 8 days.
Storage of formulations: At room temperature (between +15 and +25 °C).

Analyses
Analysis of preparations: Four samples of approximately 1 g were taken from the middle only for each formulation, including the control, used on the first day of treatment and on suitable day during the last common week of treatment for males and females (i.e. week 4).
One set of samples (2 x 1 g) was analysed at the Test Facility within the stability period using the validated method according to study AB20658. The duplicate samples (2 x 1 g) which were stored refrigerated (between +2 and +8 °C) will be destroyed at the end of the defined stability period if not required.
Duration of treatment / exposure:
Males: from 14 days before mating, throughout mating and up to the day before necropsy (28 days of exposure).
Females: from 14 days before mating, throughout mating and gestation and up to day 3 of lactation.
Frequency of treatment:
Once daily
Details on study schedule:
First day of treatment (day 0): 27 October 2014.
Mating: from 10 November 2014.
Necropsy of males: 24 November 2014,
Necropsy of females with their litter (day 4 of lactation): from 07 December 2014.
Experimental completion date (last necropsy): 15 December 2014.
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/g bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10 male and 10 female rats/dose
Control animals:
yes, concurrent vehicle
Details on study design:
Rationale for the dose selection: the dose levels were chosen based on the results of a 5-day dose range-finding study (AB20659) and a 4-week repeat dose toxicity study (AB20660) in the rat at doses up to 1000 mg/kg/day. In the absence of any toxicologically significant finding, the limit dose of 1000 mg/kg/day was selected at the high dose in the current study. The low and intermediate doses selected were 100 and 300 mg/kg/day, respectively.
Parental animals: Observations and examinations:
Morbidity/mortality, clinical signs, body weight, food consumption
Oestrous cyclicity (parental animals):
Animals were observed daily. During the treatment period, the animals were observed before and at least once after dosing to detect any clinical signs or reactions to treatment. A full clinical examination was performed at least weekly.
Litter observations:
For each litter the following data were recorded:
− number of male and female pups born (live and dead)
− external abnormalities of the pups
− number, weight and sex of pups alive on postnatal days 1 and 4.
Postmortem examinations (parental animals):
All adults were weighed at scheduled necropsy. All animals, including PND 4 pups, were submitted to a macroscopic examination for structural or pathological changes.
Postmortem examinations (offspring):
All adults were weighed at scheduled necropsy. All animals, including PND 4 pups, were submitted to a macroscopic examination for structural or pathological changes.
Statistics:
The following parameters were analysed statistically using the Provantis data acquisition system on each occasion for males and females separately:
− body weights and body weight gains
− food consumption
− copulation and fertility indices
− terminal body weights, absolute and relative organ weights.
Reproductive indices:
Pre-coital interval (in days), mating index (in %), female fertility index (in %), gestation index (in %).
Offspring viability indices:
Live birth index (in %), viability index (in %), sex ratio (proportion of male pups in %).
Clinical signs:
no effects observed
Description (incidence and severity):
There was no treatment-related clinical sign observed during the study at any dose level.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There was no unscheduled death during the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Despite some intergroup variations, there was no adverse effect of the test item on mean body weight change of the males throughout the study or for the females during the pre-mating, gestation and lactation phases.
Mean terminal body weight was consequently comparable in all groups for both sexes.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no effect of test item on mean food consumption for either sex during the pre-mating period or for the females during gestation and lactation.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
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:
no effects observed
Description (incidence and severity):
No organ weight, macroscopic or microscopic changes were noted to suggest a toxicological effect of Macrolex Rot E2G on the ovaries, testes and epididymides of treated animals.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No organ weight, macroscopic or microscopic changes were noted to suggest a toxicological effect of Macrolex Rot E2G on the ovaries, testes and epididymides of treated animals.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No organ weight, macroscopic or microscopic changes were noted to suggest a toxicological effect of Macrolex Rot E2G on the ovaries, testes and epididymides of treated animals.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
There were no adverse effects of Macrolex Rot E2G on the reproduction and/or development of the male and female rat up to the limit dose of 1000 mg/kg/day inclusive.
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
There were no adverse effects of Macrolex Rot E2G on the reproduction and/or development of the male and female rat up to the limit dose of 1000 mg/kg/day inclusive.
Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: 1000 mg/kg bw/day was the highest applied dose
Key result
Critical effects observed:
no
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
No stillborn pups were observed in any group. There was no influence of the test item on pre-birth loss in any group since the mean numbers of implantation sites and delivered pups were similar to those in the control group.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no influence of the test item on mean pup weight at birth and postnatal day 4 for either sex in any group.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no macroscopic findings at necropsy of the F1 pups associated with maternal test item exposure. Autolysis was noted in one dead pup.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There was no evidence of toxicological treatment-related findings associated with oral (gavage) administration of Macrolex Rot E2G.
Histopathological findings:
not examined
Description (incidence and severity):
There was no evidence of toxicological treatment-related findings associated with oral (gavage) administration of Macrolex Rot E2G.
Other effects:
no effects observed
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Based on the absence of treatment-related findings in the organs examined, the no observed effect level (NOEL) was set at 1000 mg/kg/day.
Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There were no adverse effects of Macrolex Rot E2G on the reproduction and/or development of the male and female rat up to the limit dose of 1000 mg/kg/day inclusive.
Remarks on result:
other: 1000 mg/kg bw/day was the highest applied dose
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

There were no unscheduled deaths or systemic clinical signs associated with the test item.

There was no effect of the test item on mean body weight change or food consumption for either sex throughout the study.

There was no adverse effect of the test item on mating performance or fertility. The mean duration of gestation was normal (approximately 22 days) in all groups. There was no influence of treatment with the test item on pre- or postnatal pup survival of either sex.

Mean pup weight at birth and postnatal day 4 was comparable with that in the experimental control.

No organ weight, macroscopic or microscopic changes were noted to suggest a toxicological effect of Macrolex Rot E2G on the ovaries, testes and epididymides of treated animals.

Conclusions:
There were no adverse effects of Macrolex Rot E2G on the reproduction and/or development of the male and female rat up to the limit dose of 1000 mg/kg/day inclusive.
Executive summary:

Three groups of 10 male and 10 female Wistar Han rats were given the test item, Macrolex Rot E2G, by daily oral (gavage) administration at dose levels of 100, 300 and 1000 mg/kg/day from 14 days before mating then for up to 4 weeks for males and throughout mating, gestation and through day 3 of lactation for females. A control group of 10 male and 10 female rats received a similar volume (l0 mL/kg) of the vehicle [1 % (w/v) Carboxymethylcellulose]. Clinical condition, body weight and food consumption of the animals were monitored throughout the study. After two weeks of treatment, one male and one female of the same group were paired for a maximum of 14 days. The females were retained throughout gestation, allowed to litter and rear their young through to postnatal day (PND) 4 and then neoropsied. Vaginal smears were taken daily for each female from the first day of pairing to verify positive copulation. Litter parameters, including the number of pups born, pup survival, sex and pup weights were reoorded up to PND 4.

The dams with their pups were necropsied on day 4 of lactation, where applicable. All animals, including PND 4 pups, were submitted to a macroscopic examination. The numbers of uterine implantations were determined and the ovaries were weighed for each female. The males were necropsied after completion of the mating period and the testes and epididymides were weighed.

Selected tissue samples were fixed and preserved from all animals. Selected organs/tissues from group 1 and 4 animals were examined histopathologically.

There were no unscheduled deaths or systemic clinical signs associated with the test item.

There was no effect of the test item on mean body weight change or food consumption for either sex throughout the study.

There was no adverse effect of the test item on mating performance or fertility. The mean duration of gestation was normal (approximately 22 days) in all groups. There was no influence of treatment with the test item on pre- or postnatal pup survival of either sex.

Mean pup weight at birth and postnatal day 4 was comparable with that in the experimental control.

No organ weight, macroscopic or microscopic changes were noted to suggest a toxicological effect of Macrolex Rot E2G on the ovaries, testes and epididymides of treated animals.

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:
GLP guideline study
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

There were no unscheduled deaths or systemic clinical signs associated with the test item.

There was no effect of the test item on mean body weight change or food consumption for either sex throughout the study.

There was no adverse effect of the test item on mating performance or fertility. The mean duration of gestation was normal (approximately 22 days) in all groups. There was no influence of treatment with the test item on pre- or postnatal pup survival of either sex.

Mean pup weight at birth and postnatal day 4 was comparable with that in the experimental control.

No organ weight, macroscopic or microscopic changes were noted to suggest a toxicological effect of Macrolex Rot E2G on the ovaries, testes and epididymides of treated animals.

Effects on developmental toxicity

Description of key information

The toxicity of the test item Macrolex Rot E2G (CAS-No. 6829-22-7) on reproduction and/or development of the male and female rat was investigated in a reproduction/developmental toxicity screening test according to OECD guideline 421.

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:
GLP guideline study
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

There were no unscheduled deaths or systemic clinical signs associated with the test item.

There was no effect of the test item on mean body weight change or food consumption for either sex throughout the study.

There was no adverse effect of the test item on mating performance or fertility. The mean duration of gestation was normal (approximately 22 days) in all groups. There was no influence of treatment with the test item on pre- or postnatal pup survival of either sex.

Mean pup weight at birth and postnatal day 4 was comparable with that in the experimental control.

No organ weight, macroscopic or microscopic changes were noted to suggest a toxicological effect of Macrolex Rot E2G on the ovaries, testes and epididymides of treated animals.

Toxicity to reproduction: other studies

Description of key information

No data

Additional information

No data

Justification for classification or non-classification

No adverse effects of Macrolex Rot E2G on the reproduction and/or development of the male and female rat up to the limit dose of 1000 mg/kg/day inclusive were found in a reproduction/ developmental toxicity screening test according to OECD guideline 421.

According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is not justified.