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

Endpoint summary

Description of key information

Oral: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD TG 422, GLP): NOAEL (m) = 92.17 mg/kg bw/day / NOAEL (f) = 96.63 mg/kg bw/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: oral, other

Remarks:

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

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:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

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 870.3050

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

DETAILS OF FOOD AND WATER QUALITY:
Each batch of diet is supplied with a certificate of analysis which is verified and authorized for release by a veterinarian.
Water is analysed twice a year for chemical and bacterial contaminants.

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

Details on route of administration:

Oral (dietary admixture)
Admixture in powdered diet

Vehicle:

other: Basic powdered diet

Details on oral 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

Observations and examinations performed and frequency:

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

Clinical signs: Animals were observed daily. During the treatment period, the animals were observed twice daily (at least 4 hours apart) to detect any clinical signs or reactions to treatment. A full clinical examination was performed at 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: Food consumption for males was measured weekly for each cage of animals during pre-mating period and reported in g/animal/day.
Food consumption of females was recorded as follows and reported in g/animal/day:
− 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: The first five males per group and at least the first five females that gave birth and had live pups at L 4.
− 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).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table1)

HISTOPATHOLOGY: Yes (see table 1)

Other examinations:

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 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.

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.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Consistent with treatment-related effects on food consumption and body weight gain (see sections 11.4 and 11.5) 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:

effects observed, non-treatment-related

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):

Serum clinical chemistry: 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

Description (incidence and severity):

In the liver, minimal or slight hepatocyte hypertrophy occurred in 7 of the 10 animals at 15 000 ppm and in 4 females given 5 000 ppm. This is a well-known adaptive change to administration of a foreign compound, and was not considered adverse. Liver weights were higher than controls at all doses in males and at 5 000 and 15 000 ppm in females.
The kidneys of males at 5 000 and 15 000 ppm exhibited cortical tubular degeneration/regeneration consistent with alpha 2u-globulin nephropathy, which is well known not to be relevant to humans. It was, however, considered adverse for the rats in this study. Kidney weight was increased as a result of this change only at 15 000 ppm.In the thymus, minimal to moderate atrophy occurred in one male and three females at 15 000 ppm. This was considered likely to be secondary to body weight loss seen at this dose. Absolute and relative thymus weights were lower than controls in males and females at 15 000 ppm.
In the spleen, extramedullary haematopoiesis was increased in none of the females at 15 000 ppm. This correlated with a lower spleen weight in females at this dose only. It is considered likely that this was due to stress. This was not considered an adverse effect.
See Table 2.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In the adrenals, minimal or slight zona fasciculata vacuolation occurred in four of the five males at 15 000 ppm. This was not considered an adverse change, and it was considered likely that this
was a secondary effect of stress.

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:

no effects observed

Description (incidence and severity):

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.

Details on results:

There was no mortality in any group.
There was no treatment-related clinical signs.
There were reductions of mean body weight gain during the first week of treatment in the 5 000 and 15 000 ppm groups resulting in a reduced terminal body weight on day 28 for males from these groups. There was also reductions of mean body weight gain during gestation in all dose female groups. Thereafter, during the 4 days of lactation, mean body weight gain was comparable with, or superior to, that in the control and terminal mean body weight for females was lower than control only in the 15 000 ppm group.
There was a dose-related reduction in mean food consumption in the 5 000 and 15 000 ppm groups, in both sexes, and in the 1 500 ppm group for females, throughout the pre-mating period.
There was no obvious effect of treatment on food consumption in the 1 500 ppm group during the pre-mating period for males.
Therafter, there was a treatment-related reduction in mean food consumption in females at all dose levels throughout the gestation period and the four days of lactation.
There was no influence of treatment in any group on the functional activity (auditory reflex, pupillary reflex, righting reflex, grip strength and locomotor activity) of the treated males or females.
There was no adverse effect of treatment on clinical laboratory parameters.
At necropsy for the adut animals, no obvious effect of treatment was observed on reproductive organs/tissues weights.
Overall, at 15 000 ppm there was hepatocyte hypertrophy and increased weight in the liver of both sexes, cortical tubular changes and increased weight in the kidney 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 and there were cortical tubular changes in the kidneys in males. At 1 500 ppm, liver weights were higher than controls in males. The only adverse changes were the cortical tubular changes in the kidneys and the no adverse effect level was 1 500 ppm.

Key result

Dose descriptor:

NOAEL

Effect level:

1 500 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

food consumption and compound intake

gross pathology

Key result

Dose descriptor:

NOAEL

Effect level:

15 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reproduction

Key result

Dose descriptor:

NOAEL

Based on:

test mat.

Sex:

female

Remarks on result:

not determinable

Key result

Dose descriptor:

NOAEL

Effect level:

5 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Development

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

Text Table 2. Noteworthy Organ Weight Differences, (% differences from controls in relative weights); Main phase
Dosage (ppm):	Males			Females
	1 500	5 000	15 000	1500	5000	15000
Liver	+9	+26	+21	+2	+16	+32
Kidney	+3	+6	+23	=	=	+7
Thymus	=	+1	-13	+6	-5	-46
Spleen	-1	+8	-9	-9	-4	-22
=: less than 1 % difference.

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: post-natal 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: post-natal day

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

Conclusions:

Parental findings: the dose of 1 500 ppm is the No-Observed-Adverse-Effect-Level for males only based on body weight, food consumption and pathology changes in the 5000 and 15 000 ppm groups.
In view of the body weight, food consumption and pathology findings in all dose groups, a maternal No-Observed-Adverse-Effect-Level was not established in this study.
The severity of the findings in the 15 000 ppm group which led to lower terminal mean body weight (-18 % and -17 % for the males and females, respectively) was in excess of a maximum tolerated dose.
Pathology findings: 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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

92.17 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, conducted according to OECD TG 422. The study is thus sufficient to fulfil the standard information requirements set out in Annexes VIII - X, 8.6.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The repeated dose toxicity after oral administration of cis-Jasmone 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 (Repeated dose toxicity: oral_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 mortality in any group and no treatment-related clinical signs were observed during the whole study period for both, males and females. However, the test substance had a significant impact on the food consumption as there was a dose-related reduction in mean food consumption in the 5000 and 15000 ppm groups, in both sexes, and in the 1500 ppm group for females, throughout the pre-mating period. A treatment-related reduction in mean food consumption in females at all dose levels was also observed throughout the gestation period and the four days of lactation. As a consequence of the significantly reduced food intake, the mean body weight gain during the first week of treatment was reduced in the 5000 and 15000 ppm groups resulting in a reduced terminal body weight on day 28 for males from these groups. There was also a significant reduction of mean body weight gain during gestation in all dosed females. During the 4 days of lactation, mean body weight gain was comparable or superior to that in the control group and terminal mean body weights for females were lower when compared to the control in the 15000 ppm group only. Moreover, the test item did not affect the functional activity of the treated animals and there was no adverse effect on clinical laboratory parameters. At necropsy, no obvious treatment-related effect was observed on reproductive organs/tissues weights in adult animals. However, at 15000 ppm there was hepatocyte hypertrophy and an increased weight of the liver in both sexes, cortical tubular changes and increased weight of the kidneys 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. Effects observed at 5000 ppm included hepatocyte hypertrophy and increased liver weights when compared to controls in both sexes and additional cortical tubular changes in the kidneys in males. Finally, at 1500 ppm, liver weights were higher than controls in male animals only. Since the effects in liver were adaptative and not seen as adverse, the only adverse effect refers to the findings in the kidney of male rats. Based on body weight and weight gain as well as food consumption and gross pathology findings, the No-Observed-Adverse-Effect-Level (NOAEL) for parental/systemic toxicity was established at 1500 ppm, corresponding to 92.17 mg/kg bw/day for males and 96.63 mg/kg bw/day for females. The Since adverse effects have been detected in the mid dose group, the Lowest-Observed-Adverse-Effect-Level (LOAEL) was determined as 5000 ppm, corresponding to 284.24 mg/kg bw/day for males and 322.68 mg/kg bw/day for females.

Conversion of ppm values into mg/kg bw/day doses

In the study report of the Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test according to OECD TG 422, effect levels are referred to in ppm as the test item was administered in the diet. In order to convert the ppm values into mg/kg bw/day doses, the average food consumption (in g/animal/day) was multiplied with the respective ppm values to derive the amount of test item consumed per animal and day (mg test item/animal/day). The amount of test item consumed was then divided by the applicable animal weights. The following mean food consumption parameters and animal weights were used to calculate the mg/kg bw/day doses.

Males

mean food consumption days 0 - 13 (relative to start of study)

mean body weights at day 13 (relative to start of study)

 

Females

pre-mating period	gestation period	lactaction period
mean food consumption days 0 - 13 (relative to start of study)	mean food consumption days 0 - 20 (relative to mating)	mean food consumption days 0 - 4 (relative to littering)
mean body weights at day 13 (relative to start of study)	mean body weights at day 20 (relative to mating)	mean body weights at day 4 (relative to littering)

All data regarding mean food consumption in the different study periods (pre-mating, gestation and lactation) and regarding animal weights are summarised in the study report: pp. 55 and 79 for males; pp. 61 and 83 for females, pre-mating period; pp. 67 and 87 for females, gestation period; pp. 73 and 91 for females, lactation period. In order to follow a conservative approach, in case of No-Observed-Adverse-Effect-Levels (NOAEL) for female animals, the lowest calculated mg/kg bw/day dose was taken (chosen from the 3 NOAELs calculated for the pre-mating, gestation and lactation period).

No-Observed-Adverse-Effect-Levels (NOAEL) in ppm (as stated in study report) and mg/kg bw/day (as converted):

Males

	ppm (as stated in study report)	mg/kg bw/day (converted)
Parental (systemic) toxicity	1500	92.17
Reproduction	15000	719.44
Development	5000	284.24

 

Females

	ppm (as stated in study report)	mg/kg bw/day (converted)
Parental (systemic) toxicity	1500	96.63
Reproduction	15000	762.59
Development	5000	322.68

Additional information can be found in the attached pdf document "Conversion of ppm into mg per kg doses" attached to this endpoint summary.

Justification for classification or non-classification

The available data on repeated dose toxicity are suitable to derive a classification and labelling. The available data do not meet any criteria for classification according to Regulation (EC) No. 1272/2008 (CLP) and are therefore conclusive but not sufficient for classification.