3-methyl-2-pent-2-enylcyclopent-2-enone

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Oral: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD TG 422, GLP): NOAEL reproduction = 15000 ppm (corresponding to 719.44 mg/kg bw/day for males and 762.59 mg/kg bw/day for females)

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 July 2014 - 29 November 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

- Vehicle (basic powdered diet) was stored between +15 and +23 °C before preparation
instead of +10 and +16 °C.
− The spare animals were euthanized and not returned to stock (error in the study plan).
− In the DRF phase, the age of the animals at the start of treatment was approximately 9 to 10 weeks old instead of 8 to 9 weeks old (supply error). Cellulose bedding (Serlab,Montataire, France), analysed at least twice a year for chemical and bacterial contaminants, was also used as bedding at the end of the gestation period and during the lactation period.
− Wooden gnawing block was not distributed to animals (error in the study plan).
− There is no certificate of analysis for Serlab cellulose bedding.
− On several occasions (most of them during the weekend), there was less than 4 hours between the two daily clinical observations, see section 5.2 for details.
- Only forelimb grip strength was performed for males instead of both forelimb and hindlimb, in error.
− Number, weight and sex of pups alive was recorded on day 1 and on day 4 post-partum and not on day 1 to day 4 post-partum.
− Tissue preparation and microscopic examination were performed for the salivary glands and not for the rectum (error in the organ processing table presented in the study plan).
- Missing information in the study plan: Open field test and pre-coital interval data were analysed using a SAS software package. Levene’s test was used to test the equality of variance across groups and Shapiro-Wilk’s test was used to assess the normality of the data distribution in each group. Data with homogenous variances and normal distribution in all groups were analysed using Anova followed by Dunnett’s test. Data showing non-homogenous variances or a non-normal distribution in at least one group were analysed using Kruskal-Wallis test followed by the Wilcoxon’s rank sum test.

Deviations:

yes

Remarks:

These deviations were considered not to have affected the outcome or the achievement of the study objectives.

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650

Version / remarks:

Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000

- Vehicle (basic powdered diet) was stored between +15 and +23 °C before preparation
instead of +10 and +16 °C.
− The spare animals were euthanized and not returned to stock (error in the study plan).
− In the DRF phase, the age of the animals at the start of treatment was approximately 9 to 10 weeks old instead of 8 to 9 weeks old (supply error). Cellulose bedding (Serlab,Montataire, France), analysed at least twice a year for chemical and bacterial contaminants, was also used as bedding at the end of the gestation period and during the lactation period.
− Wooden gnawing block was not distributed to animals (error in the study plan).
− There is no certificate of analysis for Serlab cellulose bedding.
− On several occasions (most of them during the weekend), there was less than 4 hours between the two daily clinical observations, see section 5.2 for details.
- Only forelimb grip strength was performed for males instead of both forelimb and hindlimb, in error.
− Number, weight and sex of pups alive was recorded on day 1 and on day 4 post-partum and not on day 1 to day 4 post-partum.
− Tissue preparation and microscopic examination were performed for the salivary glands and not for the rectum (error in the organ processing table presented in the study plan).
- Missing information in the study plan: Open field test and pre-coital interval data were analysed using a SAS software package. Levene’s test was used to test the equality of variance across groups and Shapiro-Wilk’s test was used to assess the normality of the data distribution in each group. Data with homogenous variances and normal distribution in all groups were analysed using Anova followed by Dunnett’s test. Data showing non-homogenous variances or a non-normal distribution in at least one group were analysed using Kruskal-Wallis test followed by the Wilcoxon’s rank sum test.

Deviations:

yes

Remarks:

These deviations were considered not to have affected the outcome or the achievement of the study objectives.

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3550

Version / remarks:

Reproduction/Developmental Toxicity Screening Test, July 2000

- Vehicle (basic powdered diet) was stored between +15 and +23 °C before preparation
instead of +10 and +16 °C.
− The spare animals were euthanized and not returned to stock (error in the study plan).
− In the DRF phase, the age of the animals at the start of treatment was approximately 9 to 10 weeks old instead of 8 to 9 weeks old (supply error). Cellulose bedding (Serlab,Montataire, France), analysed at least twice a year for chemical and bacterial contaminants, was also used as bedding at the end of the gestation period and during the lactation period.
− Wooden gnawing block was not distributed to animals (error in the study plan).
− There is no certificate of analysis for Serlab cellulose bedding.
− On several occasions (most of them during the weekend), there was less than 4 hours between the two daily clinical observations, see section 5.2 for details.
- Only forelimb grip strength was performed for males instead of both forelimb and hindlimb, in error.
− Number, weight and sex of pups alive was recorded on day 1 and on day 4 post-partum and not on day 1 to day 4 post-partum.
− Tissue preparation and microscopic examination were performed for the salivary glands and not for the rectum (error in the organ processing table presented in the study plan).
- Missing information in the study plan: Open field test and pre-coital interval data were analysed using a SAS software package. Levene’s test was used to test the equality of variance across groups and Shapiro-Wilk’s test was used to assess the normality of the data distribution in each group. Data with homogenous variances and normal distribution in all groups were analysed using Anova followed by Dunnett’s test. Data showing non-homogenous variances or a non-normal distribution in at least one group were analysed using Kruskal-Wallis test followed by the Wilcoxon’s rank sum test.

Deviations:

yes

Remarks:

These deviations were considered not to have affected the outcome or the achievement of the study objectives.

Qualifier:

according to guideline

Guideline:

other: EU Method B.7

Version / remarks:

(EC) No 440/2008 Part B; B.7: "Repeated Dose (28 days) Toxicity (oral)". OJ of the EU No. L142, May 2008
- Vehicle (basic powdered diet) was stored between +15 and +23 °C before preparation
instead of +10 and +16 °C.
− The spare animals were euthanized and not returned to stock (error in the study plan).
− In the DRF phase, the age of the animals at the start of treatment was approximately 9 to 10 weeks old instead of 8 to 9 weeks old (supply error). Cellulose bedding (Serlab,Montataire, France), analysed at least twice a year for chemical and bacterial contaminants, was also used as bedding at the end of the gestation period and during the lactation period.
− Wooden gnawing block was not distributed to animals (error in the study plan).
− There is no certificate of analysis for Serlab cellulose bedding.
− On several occasions (most of them during the weekend), there was less than 4 hours between the two daily clinical observations, see section 5.2 for details.
- Only forelimb grip strength was performed for males instead of both forelimb and hindlimb, in error.
− Number, weight and sex of pups alive was recorded on day 1 and on day 4 post-partum and not on day 1 to day 4 post-partum.
− Tissue preparation and microscopic examination were performed for the salivary glands and not for the rectum (error in the organ processing table presented in the study plan).
- Missing information in the study plan: Open field test and pre-coital interval data were analysed using a SAS software package. Levene’s test was used to test the equality of variance across groups and Shapiro-Wilk’s test was used to assess the normality of the data distribution in each group. Data with homogenous variances and normal distribution in all groups were analysed using Anova followed by Dunnett’s test. Data showing non-homogenous variances or a non-normal distribution in at least one group were analysed using Kruskal-Wallis test followed by the Wilcoxon’s rank sum test.

Deviations:

yes

Remarks:

These deviations were considered not to have affected the outcome or the achievement of the study objectives.

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3050

Version / remarks:

Repeated Dose 28-day oral toxicity study in rodents, July 2000

- Vehicle (basic powdered diet) was stored between +15 and +23 °C before preparation
instead of +10 and +16 °C.
− The spare animals were euthanized and not returned to stock (error in the study plan).
− In the DRF phase, the age of the animals at the start of treatment was approximately 9 to 10 weeks old instead of 8 to 9 weeks old (supply error). Cellulose bedding (Serlab,Montataire, France), analysed at least twice a year for chemical and bacterial contaminants, was also used as bedding at the end of the gestation period and during the lactation period.
− Wooden gnawing block was not distributed to animals (error in the study plan).
− There is no certificate of analysis for Serlab cellulose bedding.
− On several occasions (most of them during the weekend), there was less than 4 hours between the two daily clinical observations, see section 5.2 for details.
- Only forelimb grip strength was performed for males instead of both forelimb and hindlimb, in error.
− Number, weight and sex of pups alive was recorded on day 1 and on day 4 post-partum and not on day 1 to day 4 post-partum.
− Tissue preparation and microscopic examination were performed for the salivary glands and not for the rectum (error in the organ processing table presented in the study plan).
- Missing information in the study plan: Open field test and pre-coital interval data were analysed using a SAS software package. Levene’s test was used to test the equality of variance across groups and Shapiro-Wilk’s test was used to assess the normality of the data distribution in each group. Data with homogenous variances and normal distribution in all groups were analysed using Anova followed by Dunnett’s test. Data showing non-homogenous variances or a non-normal distribution in at least one group were analysed using Kruskal-Wallis test followed by the Wilcoxon’s rank sum test.

Deviations:

yes

Remarks:

These deviations were considered not to have affected the outcome or the achievement of the study objectives.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Crl: WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Virgin males: approximately 10 weeks. Virgin females: approximately 9 weeks.
- Weight at study initiation: Virgin males: 293 to 380 g. Virgin females: 182 to 248 g.
- Fasting period before study: approximately 16 hours before clinical laboratory blood sampling
- Housing: Cellulose bedding and dust-free sawdust. Shredded paper was provided as enrichment.
- Diet: Rat powdered complete diet ad libitum
- Water: Softened and filtered (0.2 μm) mains drinking water was available ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 + 3 °C
- Humidity: 35 to 70 %
- Air changes: At least 10 air changes per hour
- Photoperiod: 12 hours light (artificial)/12 hours dark (except when required for technical acts)

Route of administration:

oral: feed

Vehicle:

other: Basic powdered diet

Details on exposure:

For both sexes: 14 days before mating, throughout the mating period and up to the day of necropsy. The first day of dosing is designated as day 0.
For females: during gestation (the first day of gestation is designated as G 0) and for 4 days after parturition (the day of birth is designated as L 0).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

On the day of analysis the samples were, when required, defrosted at room temperature. Samples were accurately weighed into containers. The samples were extracted with n-hexane. The shaking time was 30 minutes. The solutions were filtered. The filtered phases were further diluted to obtain an end solution of n-hexane and concentrations within the calibration range.
Instrument: Gas chromatograph
Detector: Flame ionisation detector
Column: DB-5; 25 m × 320 μm i.d., df = 0.25 μm
Carrier gas: Helium
Carrier gas flow: 2.00 ml/min
Coefficient of correlation: (r) > 0.99
Procedural recovery samples: within the criterion of 80-120%
In the Group 1 diet, no test substance was detected. The diets of Group 2, Group 3 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Duration of treatment / exposure:

For both sexes: 14 days before mating, throughout the mating period and up to the day of necropsy

Frequency of treatment:

Continuously.

Dose / conc.:

0 ppm

Dose / conc.:

1 500 ppm

Dose / conc.:

5 000 ppm

Dose / conc.:

15 000 ppm

No. of animals per sex per dose:

10

Control animals:

yes, concurrent no treatment

yes, plain diet

Details on study design:

- Dose selection rationale:
- Rationale for animal assignment: computer-generated randomisation

Positive control:

None

Parental animals: Observations and examinations:

Morbidity/mortality: All adults were observed twice daily, at the beginning and at the end of each working day (including weekends and public holidays).

Clinical signs: Animals were observed daily. During the treatment period: twice daily (at least 4 hours apart), full clinical examination: least once weekly. Towards the end of the gestation, females were examined daily for signs of parturition.

Body weight: Males were weighed once weekly. Females were weighed as follows:
− once weekly during pre-mating and mating periods (only pre-mating data are reported)
− on days 0, 7, 14 and 20 of gestation
− on days 1 and 4 of lactation.

Food consumption:
- males: measured weekly during pre-mating period
- females: once weekly during pre-mating period; on days 0 to 7, 7 to 14 and 14 to 20 of gestation; on days 1 to 4 of lactation.

Functional tests:
− Males: at the end of the treatment period, shortly before euthanasia (day 28).
− Females: during lactation, shortly before euthanasia (day 4 of lactation).

Clinical laboratory determinations: Blood collection: First five animals/sex/group, at the end of the pre-mating period (day 14).

Mating: Vaginal smears were taken daily from the females during cohabitation.

Pregnancy and parturition: For each female, the following data were recorded:
− date of mating (day 0 of gestation or G 0)
− date of parturition (day 0 of lactation or L 0)
− duration of gestation
− abnormalities of delivery, nesting or nursing behaviour.

Litter observations:

Morbidity/mortality: Offspring were examined daily.

Litter data: For each litter the following data were recorded:
− number of pups born (live and dead)
− external abnormalities of the pups
− number, weight and sex of pups alive on lactation days 1 and 4.

Postmortem examinations (parental animals):

SACRIFICE:
 Males: after completion of the mating period (28 days of dietary administration).
 Females: on day 4 of lactation.
 Three females with total litter death, necropsied on days 1, 2 or 3 of lactation.

GROSS PATHOLOGY: Yes (see table1)

HISTOPATHOLOGY: Yes (see table 1)

Postmortem examinations (offspring):

- Presence of milk in their stomach
- Defects or cause of death were evaluated

Statistics:

The following parameters were analysed statistically on each occasion for males and females separately:
− body weights and body weight gains
− food consumption
− open field test
− haematology, coagulation and serum clinical chemistry parameters
− pre-coital interval, copulation and fertility indices
− terminal body weights, absolute and relative organ weights.

Reproductive indices:

Male and female copulation index (in %): ((number of inseminated females)/(number of paired animals)) x 100

Offspring viability indices:

Viability index (in %): ((number of pups alive on Postnatal day 1)/(number of pups born)) x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Consistent with treatment-related effects on food consumption and body weight gain in the 15 000 ppm group, a thin appearance developed for one female (no. 178) at the end of the pre-mating period that persisted through to day 7 of gestation and for a second female (no. 173) on day 1 of lactation. There were no other treatment related clinical signs. Scabs, hairloss or piloerection observed occasionally were considered incidental.

Mortality:

no mortality observed

Description (incidence):

There was no unscheduled death in any group during the study.
However, three females given 15 000 ppm (nos. 171, 173 and 179) were sacrificed following total litter death post partum. There were no treatment-related necropsy findings for these
females.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See Table 3: Body weight
Pre-mating and mating periods: As a consequence of reduced mean body weight gain and mean body weight loss during the first week of the study (days 0 to 7) for each sex in the 5000 and 15 000 ppm groups, respectively, absolute mean body weight was statistically significantly lower in each of these groups compared with the control through to termination for the males (-8 % and -18 %, respectively) and at the end of the pre-mating period for females in the 15 000 ppm group only (-8 %). There were no effect of treatment on mean body weight gain for either sex in the 1 500 ppm group.
Gestation period: Overall mean body weight gain during gestation (from G 0 to G 20) was statistically significantly lower in all treated groups ( -16 %, -17 % and -37 % in the 1 500, 5 000 and 15 000 ppm groups, respectively) when compared with control. Absolute mean body weight on G 20 was consequently statistically significantly lower in each of the treated groups (-8 %, -8 % and -20 % in the 1 500, 5 000 and 15 000 ppm groups, respectively) when compared with control.
Lactation period: Mean body weight gain was comparable in all groups during days 1 to 4 of lactation. However, absolute mean body weight tended to remain lower in all treated groups but the difference from the control only attained statistical significance in the 15 000 ppm group through to termination (-19 %).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

See Table 4: Food consumption
Pre-mating period: On average, there was a dose-related and statistically significant reduction in mean food consumption for both sexes in the 5 000 and 15 000 ppm groups and for the females only in the 1 500 ppm group during the pre-mating period (days 0 to 13). The effect was most pronounced during the first week of dosing (Days 0 to 7) with animals consuming less than 50 % of that in the control in the high dose group. There was no obvious effect of treatment on mean food consumption for the males in the 1 500 ppm group.
Gestation period: There was a dose-related and statistically significant reduction in mean food consumption in all treated groups (on average -9 %, -10 % and -26 %, in the 1 500, 5 000 and 15 000 ppm groups, respectively) compared with the control throughout gestation (G 0 to G 20).
Lactation period: Mean food consumption remained lower in all treated groups during the lactation phase compared with the control (-15 %, -6 % and -35 %, in the 1500, 5 000 and 15 000 ppm groups, respectively) attaining statistical significance in the high dose group only.

Haematological findings:

no effects observed

Description (incidence and severity):

There was no influence of treatment on the haematology parameters for either sex in any group. The total white blood cell count was slightly lower in each of the treated groups compared with the concurrent control principally due to slightly (statistically significant for females only) lower neutrophil counts. However, this was due to an incidentally higher mean white blood cell count in the control group compared with the historical control data.
Coagulation: There was no influence of treatment on the coagulation parameters for either sex in any group.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The only differences from the control that suggested any association with treatment included slightly but generally statistically significantly higher mean cholesterol and triglyceride
concentrations for both sexes in the 15 000 ppm group. However, since mean values remained within the historical control ranges, the minor differences were considered to be of no
toxicological significance.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The dose of 1 500 ppm is the No-Observed-Adverse-Effect-Level based on treatment-related effects on the cortical tubular changes in the kidneys in males at 5 000 and 15 000 ppm considered adverse for the rats in this study. Other non adverse findings were observed at 15 000 ppm and consisted in hepatocyte hypertrophy and increased weight in the liver of both sexes, of males, atrophy and decreased weight (in both sexes) of the thymus, zona fasciculata vacuolation in the adrenals of males and decreased extramedullary haematopoiesis and decreased weight in the spleen of females. At 5 000 ppm, there was hepatocyte hypertrophy and liver weights were higher than controls in both sexes. At 1 500 ppm, liver weights were higher than controls in males. There were no histological findings in the reproductive organs from the three females with total litter death that could have caused the loss of the litters.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

Only non-neoplastic hispathological findings observed.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Test item-related histologic changes were noted in the liver, kidneys, thymus, adrenal and spleen at 15 000 ppm and in the liver and kidneys at 5 000 ppm.
Functional tests: There was no obvious influence of treatment on the functional tests of the treated males or females as shown by the auditory, pupillary and righting reflexes, or grip strength. Although there was some intergroup variation, there was no difference from the control and/or historical control data to suggest any adverse effect of treatment on locomotor activity as assessed in the open field test.

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no toxicologically significant differences from the control for the mating performance.
There was no treatment-related effect on mating performance in any group. All animals mated and the majority of females in each group showed evidence of insemination within the first 4 days of pairing (approximate duration of a normal estrous cycle). The mean pre-coital interval was normal (less than 4 days) in all groups.
There was no treatment-related effect on fertility of either sex in any group. All inseminated females became pregnant.
All pregnant females in each group completed delivery and the mean duration of gestation (approximately 22 days) was comparable in the control and treated groups.

There was no influence of treatment on the haematology parameters for either sex in any group.
Consistent with treatment-related effects on food consumption and body weight gain in the 15 000 ppm group.
there was a dose-related and statistically significant reduction in mean food consumption for both sexes in the 5 000 and 15 000 ppm groups and for the females only in the 1 500 ppm group during the pre-mating period (days 0 to 13).
There was no treatment-related effect on mating performance and fertility in any group.
There was no treatment-related effect on parturition or the duration of gestation at any dose level.
There was no treatment-related effect on pre-birth loss in any group.
There were no histological findings in the reproductive organs from the three females with total litter death that could have caused the loss of the litters.
Test item-related histologic changes were noted in the liver, kidneys, thymus, adrenal and spleen at 15 000 ppm and in the liver and kidneys at 5 000 ppm.

Key result

Dose descriptor:

NOAEL

Effect level:

15 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

5 000 ppm

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

All pups were noted as weak and/or cold to touch prior to death for two of the females (nos. 173 and 179) that had total litter death in the 15 000 ppm group. One pup was also noted to be weak for the third female (no. 171) with a total litter death.
Other pup observations such as cold to touch, cyanosis, weakness or constriction of the tail for a few pups in isolated litters in the low dose group and the control were considered to be
incidental.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

The live birth index was slightly lower in the 15 000 ppm group (91.2 %) due to two litters (female nos. 171 and 179) with a total of 10 stillborn pups compared with a single litter (no. 117) with 4 stillborn pups in the concurrent control (97.1 %). There was also a total of 7 stillborn pups from two litters (female nos. 137 and 140) in the 1 500 ppm group but in the absence of any similar finding in the 5 000 ppm group, this was considered to be incidental. The number of pups subsequently dead or missing was higher in the 15 000 ppm group (31 in total) compared with none in the control. Consistent with this finding, three females (nos. 171, 173 and 179) lost their litter between PND 1 and PND 4 compared with none in the control and other lower dose groups. The corresponding PND 4 viability index was therefore markedly lower in the 15 000 ppm group (69.9 %) than in the control (100 %) and the historical control data range (94.1 to 100 %). As a consequence, the mean live litter size in the 15 000 ppm group on PND 4 was markedly lower (7.2 pups per litter) than in the control (13.2). There was also a total of 7 dead or missing pups in the 1 500 ppm group but in the absence of any similar finding in the 5000 ppm group (a single dead pup), this was considered to be incidental.
The mean live litter size on PND 4 in the 1 500 and 5 000 ppm groups was comparable with the historical control data but lower than in the concurrent control due to the incidental difference in the pre-birth data.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Neither the incidence nor type of the macroscopic changes noted at necropsy of the pups at postnatal day 4, such as a dark left epididymis and testis for one pup in the 5 000 ppm group suggested any association with maternal treatment. Autolysis and cannibalisation were frequently noted amongst the stillborn/dead pups.

There was a treatment-related effect on postnatal pup survival and growth in the 15 000 ppm group only.
Necropsy examination did not reveal any treatment-related findings in the pups.
Postnatal pup viability and development was compromised in the 15 000 ppm group as shown by an increased incidence of pup mortality and reduced pup weight at birth and postnatal day 4. Consistent with these observations, three females lost their entire litters post partum. These findings are considered to be secondary to the observed maternal toxicity rather than a direct effect of the test substance on postnatal pup survival and development.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

5 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

body weight and weight gain

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

15 000 ppm

Treatment related:

no

Table 3: Body weight (P0)

Salient mean body weight differences (absolute and change in grams)

Dose group (ppm)	0	1500	5 000	15000
Males
Days 0 to 7	19.9	18.5	8.7***	-22.6***
Day 28	376.3	379.1	346.3*	309.5**
Females
Days 0 to 7	12.5	12.4	7.1*	-10.0***
Day 13	231.1	226.8	225.8	211.5#
G 0 to G 20	143.6	120.9###	119.4###	90.0###
PND 1	285.2	262.7#	267.3#	220.1###
PND 1 to PND 4	21.2	21.8	23.4	22.2
PND 4	306.3	284.5	290.6	253.2###

PND: postanal day

Shirley 2 sided test: * p<0.05; ** p<0.01; *** p < 0.001.

Williams 2 sided test: # p<0.05; ### p<0.001.

Table 4: Food consumption (P0)

Salient mean food consumption (grams/animal/day) values

Dose group (ppm)	0	1500	5 000	15000
Males
Days 0 to 7	22.7	22.1*	19.2***	15.6***
Females
Days 0 to 13	17.7	16.1***	15.2***	10.8***
G 0 to G 20	24.9	22.5##	22.4##	18.5###
PND 1 to PND 4	38.2	32.3	35.8	25.0###
PND 1 to PND 4 % of difference with control	Not applicable	-15%	-6%	-35%

PND: postanal day

Shirley 2 sided test: ** p<0.05; *** p<0.001; Williams 2 sided test: ## p<0.01; ### p<0.001.

PND: postanal day

Shirley 2 sided test: ** p<0.05; *** p<0.001; Williams 2 sided test: ## p<0.01; ### p<0.001.

Conclusions:

Reproduction findings: the dose of 15 000 ppm is the No-Observed-Adverse-Effect-Level in the absence of any adverse effect of treatment on male and female reproductive performance including gonadal function, mating behaviour and conception in any group.
Developmental findings: the dose of 5 000 ppm is the No-Observed-Adverse-Effect-Level based on treatment-related effects on early postnatal development (pup mortality and reduced pup
weight) in the 15 000 ppm group. These findings were considered to be consistent with the severity of the maternal toxicity observed in the high dose group.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

719.44 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable (Klimisch score 1) Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test according to OECD TG 422. The study is thus sufficient to fulfil the standard information requirements set out in Annexes VIII - X, 8.7.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The potential of cis-Jasmone to induce effects related to reproduction and developmental toxicity has been investigated in a combined repeated dose toxicity study with the reproduction / developmental toxicity screening test according to OECD TG 422 and under GLP conditions (Toxicity to reproduction_2015). Groups of 10 Wistar (Crl: WI(Han)) rats per sex and dose were exposed to 0, 1500, 5000 and 15000 ppm of the test substance in the diet (admixture in powdered diet). Animals of both sexes were dosed 14 days before mating, throughout the mating period and up to the day of necropsy. With respect to the females, these were also dosed during gestation and for 4 days after parturition. The animals of the control group received the plain diet. There was no unscheduled death in any group during the study. However, three females dosed with 15000 ppm were sacrificed following total litter death post partum. Upon necropsy, no histological alterations were found affecting the reproductive organs of the females with total litter death that could have caused the loss of the litters. Moreover, there was no influence of treatment on the haematology parameters for either sex in any group. Consistent with treatment-related effects on food consumption and body weight gain in the 15000 ppm group, a dose-related and statistically significant reduction in mean food consumption for both sexes in the 5000 and 15000 ppm groups and for the females only in the 1500 ppm group was observed during the pre-mating period (days 0 to 13). Test item-related histologic changes were noted in the liver, kidneys, thymus, adrenal and spleen at 15000 ppm and in the liver and kidneys at 5000 ppm. However, only those referring to male kindeys are considered as adverse effects. No treatment-related effects on the investigated reproduction and developmental parameters were detected in any animal at any dose level. Mating performance and fertility, parturition and the duration of gestation as well as pre-birth losses were normal when compared to the control animals. All animals mated and the majority of females in each group showed evidence of insemination within the first 4 days of pairing (approximate duration of a normal estrous cycle). The mean pre-coital interval was normal (less than 4 days) in all groups. All inseminated females became pregnant. All pregnant females in each group completed delivery and the mean duration of gestation (approximately 22 days) was comparable in the control and treated groups.

All pups were noted as weak and/or cold to touch prior to death for two of the females that had total litter death in the 15000 ppm group. One pup was also noted to be weak for the third female with a total litter death. Other pup observations such as cold to touch, cyanosis, weakness or constriction of the tail for a few pups in isolated litters in the low dose group and the control were considered to be incidental. The live birth index was slightly lower in the 15000 ppm group (91.2 % when compared to controls) due to two litters with a total of 10 stillborn pups compared with a single litter with 4 stillborn pups in the concurrent control (97.1 %). There was also a total of 7 stillborn pups from two litters in the 1500 ppm group but in the absence of any similar finding in the 5000 ppm group, this was considered to be incidental. The number of pups subsequently dead or missing was higher in the 15000 ppm group (31 in total) compared with none in the control. Consistent with this finding, three females of this group lost their litter between PND 1 and PND 4 compared with none in the control and other lower dose groups. The corresponding PND 4 viability index was therefore markedly lower in the 15000 ppm group (69.9 %) than in the control (100 %) and the historical control data range (94.1 to 100 %). As a consequence, the mean live litter size in the 15000 ppm group on PND 4 was significantly lower (7.2 pups per litter) than in the control (13.2). Moreover, there was also a total of 7 dead or missing pups in the 1500 ppm group but in the absence of any similar finding in the 5000 ppm group (a single dead pup), this was considered to be incidental. The mean live litter size on PND 4 in the 1500 and 5000 ppm groups was comparable with the historical control data but lower than in the concurrent control due to the incidental difference in the pre-birth data. Neither the incidence nor type of the macroscopic changes noted at necropsy of the pups at postnatal day 4, such as a dark left epididymis and testis for one pup in the 5000 ppm group suggested any association with maternal treatment. Autolysis and cannibalisation were frequently noted amongst the stillborn/dead pups.

Based on the absence of any adverse effect of treatment on male and female reproductive performance including gonadal function, mating behaviour and conception in any group, the No-Observed-Adverse-Effect-Level (NOAEL) for reproduction was determined to be 15000 ppm (corresponding to 719.44 mg/kg bw/day for male and 762.59 mg/kg bw/day for female animals. In addition, based on treatment-related effects on early postnatal development (pup mortality and reduced pup weight), the No-Observed-Adverse-Effect-Level (NOAEL) for development could be established at 5000 ppm, corresponding to 284.24 mg/kg bw/day for males and 322.68 mg/kg bw/day for females. It must be noted, however, that the effects referring to the developmental toxicity were noticed at a dose level which was clearly toxic to parental animals from a systemic point of view. Therefore, they are to be considered as secondary due to parental toxicity.

A detailed description of how ppm values given in the study report have been converted into mg/kg bw/day doses is provided in the endpoint summary Repeated dose toxicity, section 7.5.

Effects on developmental toxicity

Description of key information

Oral: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD TG 422, GLP): NOAEL development = 5000 ppm (corresponding to 284.24 mg/kg bw/day for males and 322.68 mg/kg bw/day for females)

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Dose descriptor:

NOAEL

284.24 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information consists of an adequate and reliable (Klimisch score 1) Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test. The study is thus sufficient to fulfil the standard information requirements set out in Annexes VIII - X, 8.7.

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 detailed description of relevant information derived from the Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test is summarised under 'Additional information' in the section 'Effects on fertility'.

Justification for classification or non-classification

The available data provide no sufficient evidence that would imply any classification and labeling with respect to reproductive and developmental toxicity according to Regulation (EC) No. 1272/2008 (CLP).

However, as no prenatal developmental toxicity study is available, the conclusion for classification is ‘data lacking’.