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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 December 2011 - 19 April 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted and well described study in accordance with GLP and OECD Guideline 422 without any deviation
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
other: audited draft report
Title:
Unnamed
Year:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
(1r,4r)-1-methyl-4-(propan-2-yl)cyclohexyl acetate; (1s,4s)-1-methyl-4-(propan-2-yl)cyclohexyl acetate; 2-[(1r,4r)-4-methylcyclohexyl]propan-2-yl acetate; 2-[(1s,4s)-4-methylcyclohexyl]propan-2-yl acetate
EC Number:
939-728-7
Molecular formula:
C12H22O2
IUPAC Name:
(1r,4r)-1-methyl-4-(propan-2-yl)cyclohexyl acetate; (1s,4s)-1-methyl-4-(propan-2-yl)cyclohexyl acetate; 2-[(1r,4r)-4-methylcyclohexyl]propan-2-yl acetate; 2-[(1s,4s)-4-methylcyclohexyl]propan-2-yl acetate
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Menthanyl acetate multiconstituent
- Physical state: Colourless - slightly amber liquid
- Analytical purity: 98.4 % (sum of the main constituents >10 %)
- Lot/batch No.: 120922
- Date received: 09 November 2011
- Manufacture date of the lot/batch: 05 July 2011
- Expiration date of the lot/batch: 04 July 2013
- Storage condition of test material: Approximately 4 ºC in the dark, under nitrogen

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, UK
- Age at study initiation: Approximately 10 weeks
- Weight at study initiation: Males: 363-448 g; females: 218-283 g
- Housing: All animals during the pre-mating phase, all mated males, recovery group females and toxicity phase females were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids. During the mating phase, one male and one female within each dose group were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper. Mated females were housed individually during gestation and lactation, in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet (e.g. ad libitum): Ground diet (Rodent PMI 5002 (Certified), BCM IPS Limited, London, UK), ad libitum
- Water (e.g. ad libitum): Mains drinking water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 2 °C
- Humidity: 55 ± 15 %
- Air changes: At least 15 air changes/h
- Photoperiod: 12 h dark / 12 h fluorescent light

IN-LIFE DATES: From: 17 February 2012 To: 19 April 2012

Administration / exposure

Route of administration:
oral: feed
Vehicle:
corn oil
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Dietary admixtures were prepared prior to the first treatment, and twice monthly thereafter.
- Mixing appropriate amounts with (Type of food): A known amount of test item was initially mixed with 2 % corn oil and subsequently a small amount of basal laboratory diet was incorporated until homogeneous in a Robot Coupe Blixer 4 set at a constant speed. This pre-mix was then added to a larger amount of basal laboratory diet and mixed for a further thirty minutes at a constant speed, setting 1, in a Hobart H800 mixer.
- Storage temperature of food: Diet was stored in labelled, double plastic bags in labelled, covered plastic bins at room temperature.
- Stability: Dietary admixtures were found to be stable for 26 days at room temperature.
Details on mating procedure:
- M/F ratio per cage: 1: 1
- Length of cohabitation: Up to 14 days
- Proof of pregnancy: Presence of sperm within the vaginal smear and/or vaginal plug referred to as Day 0 of pregnancy
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Method: The concentration of test item in the dietary admixtures was determined by gas chromatography (GC) using an external standard technique.
- Conditions: GC system: Agilent Technologies 5890, incorporating autosampler and workstation; Column: DB-5 (30 m x 0.53 mm id x 5 µm film)
- Sampling: A representative sample of the dietary admixtures was accurately weighed and extracted in a suitable volume of acetonitrile. The extract was diluted using acetonitrile to give a final, theoretical test item concentration of approximately 100 mg/kg.
- Results: The mean concentrations achieved for each dose mix were within specification at a range of 94-98 %.
Duration of treatment / exposure:
- Main phase: Males were dosed daily during premating and mating periods and up to 42 days; females were dosed up to 63 consecutive days (including a three week maturation phase, pairing, gestation and early lactation).
- Toxicity phase: 42 days
- Recovery phase: Treated for 42 days and then maintained without treatment for a further 14 days
Frequency of treatment:
Once daily
Details on study schedule:
None
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 800, 1500 and 5000 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0, 50.7, 93.7 and 298.8 mg/kg bw/day
Basis:
other: equivalent to mean achieved dosages
No. of animals per sex per dose:
Main phase: 10 males and 10 females/dose (except for males from control and top dose groups: 5 males/dose)
Toxicity phase: 5 females/dose
Recovery phase: 5 rats/sex/dose at 0 and 5000 ppm
Control animals:
other: basal laboratory diet with 2 % corn oil added
Details on study design:
- Dose selection rationale: Dose levels were chosen based on the results of previous toxicity work (Project Number 41103354).
- Rationale for animal assignment: Animals were allocated to dose groups using a randomisation procedure based on stratified body weights.
Positive control:
No

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily
- Cage side observations: Overt signs of toxicity, ill-health and behavioural change

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were performed on all test and control group animals before the first exposure to the test item and for main phase males, toxicity phase females and recovery animals once weekly thereafter. Observations were also performed on main phase females weekly during the pre-mating phase and then on Days 0, 6, 13 and 20 post coitum and on Days 1 and 7 of lactation. Functional performance tests were also performed in the first five main phase males per dose group and in toxicity phase females once during the final week of treatment.
- Parameters of behavioural assessments: gait, hyper/hypothermia, tremors, skin colour, twitches, respiration, convulsions, palpebral closure, bizarre/abnormal/stereotypic behaviour, urination, salivation, defecation, pilo-erection, transfer arousal, exophthalmia, tail elevation and lachrymation
- Parameters of functional performance tests: motor activity and forelimb/hindlimb grip strength

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule: Once during the final week of treatment extensive functional observations were performed on five selected main phase males from each dose group and for all (five) toxicity females.
- Parameters: Sensory reactivity: grasp response, touch escape, vocalisation, pupil reflex, toe pinch, blink reflex, tail pinch, startle reflex and finger approach

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 and then weekly for main phase males and toxicity phase females until termination. For main phase females, individual body weights were recorded on Day 1 and then weekly until pairing. Mated females were weighed on Days 0, 6, 13 and 20 post coitum and on Days 1, 4 and 7 post-partum. Recovery animals were weighed on Day 1 and then weekly until termination.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded on Days 0-6, 6-13 and 13-20 post-coitum. For females with live litters, food consumption was recorded on Days 1, 4 and 7 post-partum. Weekly food consumption values were calculated for each cage of toxicity phase females and recovery group females throughout the study period. Weekly food consumption values were calculated for recovery group males during the pre-pairing period, after the mating phase and during the recovery period.

FOOD EFFICIENCY: Yes
- Time schedule for examinations: Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for main phase and recovery males prior to and after pairing, for toxicity and recovery phase females during the recovery period where applicable, and for main phase females prior to pairing. Due to offspring growth and milk production, food efficiency could not be accurately calculated for females, during gestation and lactation.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Daily

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Main phase males and toxicity phase females (5/dose): Day 42; Recovery phase (all animals): Day 56
- Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were taken by cardiac puncture at termination
- Animals fasted: No
- Haematology parameters: Haemoglobin, erythrocyte count (RBC), haematocrit, erythrocyte indices [mean corpuscular haemoglobin (MCH), mean corpuscular volume (mcv), mean corpuscular haemoglobin concentration (MCHC)], total leucocyte count (WBC), differential leucocyte count [neutrophils, lymphocytes, monocytes, eosinophils, basophils], platelet count, reticulocyte count, prothrombin time and activated partial thromboplastin time
- Blood Chemistry parameters: Urea, glucose, total protein, albumin, albumin/globulin ratio, sodium, potassium, chloride, calcium, inorganic phosphorus, gamma glutamyl transpeptidase, aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), alkaline phosphatase (AP), creatinine, total cholesterol, total bilirubin and bile acids

PREGNANCY AND PARTURITION:
- Each pregnant female was observed two to three times around the expected date of parturition, and following parameters were noted: date of pairing, date of mating, date and time of observed start of parturition, and date and time of observed completion of parturition.
Oestrous cyclicity (parental animals):
No data
Sperm parameters (parental animals):
No data
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- Number of offspring born
- Number of offspring alive; recorded daily and reported on Days 1, 4 and 7 post-partum
- Sex of offspring on Days 1, 4 and 7 post-partum
- Clinical condition of offspring from birth to Day 7 post-partum
- Individual offspring weights on Days 1, 4 and 7 post-partum (litter weights were calculated retrospectively from this data)
Postmortem examinations (parental animals):
SACRIFICE
- Adult main phase males and toxicity phase females were killed by intravenous overdose of a barbiturate agent followed by exsanguination on Day 43.
- Adult main phase females and recovery group animals were killed in a similar manner on Day 7 post-partum and on Day 57, respectively.

GROSS NECROPSY
- All adult animals, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
- For all main phase females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
- The following organs, removed from main phase males, toxicity phase females and recovery phase animals that were killed at the end of the study, were dissected free from fat and weighed before fixation: adrenals, brain, epididymides (left and right), heart, kidneys (left and right), liver, lungs, ovaries (left and right), pituitary, prostate, seminal vesicles, spleen, testes (left and right), thymus, thyroid (weighed post-fixation with parathyroid) and uterus (weighed with cervix and oviducts).
- The following organs, removed from main phase females that were killed at the end of the study, were dissected free from fat and weighed before fixation: ovaries (left and right) and uterus (weighted with cervix and oviducts).
- Samples of the tissues listed in table 7.8.1/1 were removed from main phase males, toxicity phase females and recovery animals and preserved in buffered 10 % formalin, except where stated.
Postmortem examinations (offspring):
SACRIFICE:
- Surviving offspring were terminated via intracardiac overdose of a barbiturate agent.

GROSS NECROPSY
- All offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
Statistics:
- Body weight, food and water consumption during gestation and lactation, pre-coital interval and gestation length, litter size and weights, sex ratio, implantation sites, implantation loss and viability indices, offspring body weight and change, haematology, blood chemistry, adult absolute and body weight-relative organ weights were subjected for statistical analysis.
- Data for males and females prior to pairing and functional performance test data were analysed by the Provantis™ Tables and Statistics Module. For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA and ANCOVA and Bartletts’s test. The transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found, but the data showed non-homogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or Steel (non-parametric) test to determine significant differences from the control group. Finally, if required, pair wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
- Data for females during gestation and lactation, and offspring data were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating Levene’s test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett’s test. Where Levene’s test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney U test.
- Non-parametric methods were used to analyse implantation loss, offspring sex ratio and landmark developmental markers.
Reproductive indices:
Mating Performance and Fertility:
- Pre-coital Interval: Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
- Mating Index (%) = (number of animals mated / number of animals paired) * 100
- Pregnancy Index (%) = (number of pregnant females / number of animals mated) * 100

Gestation and Parturition Data
- Gestation Length: Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
- Parturition Index (%) = (number of females delivering live offspring / number of pregnant females) * 100
Offspring viability indices:
- Post–implantation loss: [(number of implantation sites - total number of offspring born) / Number of implantation sites] * 100
- Live birth index (%): (number of offspring alive on Day 1 / number of offspring born) * 100
- Viability index (%): (number of offspring alive on Day 4 / number of offspring alive on Day 1) * 100
- Lactation index (%): (number of offspring alive on Day 7 / number of offspring alive on Day 1) * 100
- Sex Ratio (% males): (number of male offspring / total number of offspring) * 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
some effects were noted but considered as non-indicative of a hazard to human health
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- No unscheduled deaths or treatment-related clinical signs were noted. One main phase male treated with 5000 ppm had red stained urine on Days 15 and 20. One control main phase female had a mass on the left side of the thoracic region from Day 47 onwards whilst one recovery control male had generalised scab formation between Days 36 and 46. In the absence of treatment these were considered isolated finding of no toxicological importance. One main phase female treated with 800 ppm and one main phase control female had fur loss during the lactation period. One toxicity phase female treated with 5000 ppm also had fur loss between Days 36 and 43. Observations of this nature are commonly observed during the lactation period or in group housed animals and are considered not to be of toxicological significance.
- No treatment-related effects were noted on behavioural, sensory reactivity and functional performance parameters.

BODY WEIGHT (PARENTAL ANIMALS)
- Main phase females treated with 5000 ppm showed a significant reduction (-65 %) in body weight gain during maturation (first week of treatment) when compared to control. Recovery was evident thereafter.
- No such effects were detected in main phase males or toxicity phase females treated with 5000 ppm or animals of either sex from any phase treated with 1500 or 800 ppm.

FOOD CONSUMPTION (PARENTAL ANIMALS)
- Main phase females treated with 5000 ppm showed a slight reduction (-14 %) in food consumption during maturation. This was considered to reflect on initial reluctance to eat the diet admixture due to its low palatability.
- No such effects were detected in main phase males or toxicity phase females treated with 5000 ppm or animals of either sex from any phase treated with 1500 or 800 ppm.

FOOD EFFICIENCY (PARENTAL ANIMALS)
- Main phase females treated with 5000 ppm showed a slight reduction in food efficiency during maturation.
- No such effects were detected in main phase males or toxicity phase females treated with 5000 ppm or animals of either sex from any phase treated with 1500 or 800 ppm.

WATER CONSUMPTION (PARENTAL ANIMALS)
- Daily visual inspection of water bottles did not reveal any significant intergroup differences.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
- Mean achieved dosages for animals of either sex at 800, 1500 and 5000 ppm were 50.7, 93.7 and 298.8 mg/kg bw/day, respectively.
- Mean weekly achieved dosage was fairly consistent fairly consistent however in males it was slightly lower (5.6 times) than the anticipated 6.3 fold interval between the low and high dose levels throughout the treatment period.
- Mean achieved dosage during gestation and lactation was increased when compared to the maturation phase for main phase females. This increase was to be expected due to the increased physical demand of pregnancy and nursing of offspring.

HAEMATOLOGY (PARENTAL ANIMALS)
- No treatment-related significant changes were detected.
- Main phase males treated with 5000 ppm showed statistically significant reductions in haemoglobin, erythrocyte count, haematocrit, prothrombin time and activated partial thromboplastin time. Main phase males treated with 1500 ppm also showed a statistically significant reduction in prothrombin time. Four out of the five individual values for haemoglobin, erythrocyte count and haematocrit and all of the individual values for prothrombin time (5000 ppm and 1500 ppm dose groups) and activated partial thromboplastin time were within the normal ranges for rats of the strain and age used, therefore the intergroup differences were considered to be of no toxicological significance. Recovery group females treated with 5000 ppm showed a statistically significant increase in prothrombin time following fourteen days without treatment. In the absence of similar findings detected in toxicity phase females at the end of the treatment period the intergroup difference was considered to be of no toxicological significance.

BLOOD CHEMISTRY (PARENTAL ANIMALS)
- No treatment-related significant changes were detected.
- Main phase males from all treatment groups showed a statistically significant reduction in aspartate aminotransferase. All individual values were within the normal range for rats of the strain and age used, and in the absence of a true dose related response the intergroup differences were considered to be of no toxicological significance. Main phase males treated with 800 ppm showed a statically significant increase in urea. In the absence of a true dose related response the intergroup difference was considered to be of no toxicological significance.
- Toxicity phase females treated with 5000 ppm showed a statistically significant increase in phosphorus and a statistically significant reduction in creatinine. All individual values were within the normal range for rats of the strain and age used, therefore the intergroup differences were considered to be of no toxicological significance.
- Following 14 days without treatment the recovery group 5000 ppm males showed statistically significant increases in total protein, albumin and creatinine and statistically significant reductions in alanine aminotransferase and potassium concentration. Recovery group 5000 ppm females also showed statistically significant reductions in alanine aminotransferase and cholesterol. In the absence of similar findings detected in main phase males or toxicity phase females at the end of the treatment period the intergroup differences were considered to be of no toxicological significance.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- Mating: No treatment-related effects were detected in mating performance.
- Fertility: No treatment-related effects on fertility were detected.
- Gestation Lengths: No treatment-related effects were detected in the length of gestation between control and treated groups.

ORGAN WEIGHTS (PARENTAL ANIMALS)
- Main phase males from all treatment groups showed an increase in kidney and liver weight both absolute and relative to terminal body weight. A true dose related response however was not evident.
- Recovery males treated with 5000 ppm also showed an increase in absolute and relative liver weight. Recovery group 5000 ppm females showed a statistically significant increase in absolute and relative ovary weight. In the absence of similar effects detected in animals at the end of the treatment period or any histopathological correlates the intergroup differences were considered not to be of toxicological significance.
- Toxicity phase females treated with 5000 ppm showed an increase in liver weight both absolute and relative to terminal body weight. No such effects were detected in toxicity phase females treated with 1500 or 800 ppm.

GROSS PATHOLOGY (PARENTAL ANIMALS)
- There was no adult macroscopic finding considered to be related to test item toxicity.
- Post mortem examinations did not reveal any treatment-related macroscopic findings for interim death or terminal kill offspring.
- One main phase male treated with 5000 ppm had an enlarged left testis at necropsy, correlating histopathologically with diffuse marked unilateral tubular atrophy with slight tubular dilation. A further male from this treatment group had increased pelvic space in the left kidney, correlating with pelvic dilation histopathologically. These findings were considered to be within the range of normal background alterations and were considered not to be of toxicological importance. One recovery group 5000 ppm male had small and flaccid testes and small epididymides at necropsy. In the absence of similar effects detected in animals at the end of the treatment period, the incidental findings were considered not to be of toxicological significance. One control main phase female had a mass in the left side of the thoracic region. This was confirmed histopathologically to be an adenocarcinoma however due to the presence of the mass being in a control animal it was unrelated to treatment.

HISTOPATHOLOGY (PARENTAL ANIMALS)
The following treatment related microscopic abnormalities were detected:
- Liver: Minimal to slight centrilobular hepatocellular hypertrophy was evident in some main phase males at 1500 ppm and in all main phase males at 5000 ppm. Minimal centrilobular hypertrophy was also present in one toxicity phase female at 1500 ppm and minimal diffuse hepatocellular hypertrophy was evident in all toxicity phase females at 5000 ppm. The liver cell hypertrophy was fully reversible in recovery animals following fourteen days without treatment. Hepatocyte enlargement is commonly observed in the rodent liver following the administration of xenobiotics and in the absence of associated inflammatory or degenerative changes, is generally considered to be adaptive in nature and does not represent an adverse health effect.
- Thyroid: Minimal to slight diffuse follicular cell hypertrophy was evident in 3/5 main phase males at 5000 ppm and at minimal severity in 4/5 toxicity phase females at 5000 ppm. The thyroid follicular cell hypertrophy was fully reversible in recovery animals following 14 days without treatment. These changes are generally regarded as adaptive in nature and of limited human relevance.
- Kidneys: Slight focal and unilateral tubular degeneration/regeneration was evident in one main phase male at 5000 ppm associated with minimal tubular dilation. Hyaline droplet severity was minimally increased at 800 and 1500 ppm but at 5000 ppm was comparable to controls. Minimal focal to multifocal and unilateral to bilateral corticomedullary mineralization was evident in 4/5 toxicity phase females at 5000 ppm. Minimal focal and unilateral tubular degeneration/regeneration in the renal proximal tubules was also evident in one main phase male at 800 and 1500 ppm and minimal focal and unilateral corticomedullay mineralization was also evident in one toxicity phase female at 800 and 1500 ppm. In recovery 5000 ppm animals these kidney findings were fully or partially reversible and minimal findings were evident in one control recovery male and in two recovery control females. Therefore, the single minimal findings at 1500 and 800 ppm were considered to be incidental in the main phase male and toxicity phase females. In main phase controls and all treated main phase males, minimal to slight focal to multifocal basophilic foci were present which are considered incidental. The kidney findings present in males are likely to represent a possible atypical and very minimal variant of alpha 2u-globulin nephropathy however possible premature stages of chronic progressive nephropathy (CPN) cannot be excluded. The minimally increased corticomedullary mineralisation in females can also be a minimally exacerbated part of CPN as often seen in older females. Both findings, alpha 2u-globulin and premature or exacerbated CPN are considered to be adverse in rats but are considered to be of no relevance to man.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
298.8 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Remarks:
reproductive and developmental toxicity
Effect level:
298.8 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects were noted on mating, fertility, gestation lengths, offspring litter size, sex ratio, viability, growth and development

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

OFFSPRING LITTER SIZE, SEX RATIO AND VIABILITY
- No treatment related effects were detected in litter size or litter viability for litters from treated females compared to controls. There were no differences in sex ratio for litters from treated females when compared to litters from control females.

OFFSPRING GROWTH AND DEVELOPMENT
- Offspring bodyweight gain at birth and subsequently on Day 1, 4 and 7 post-partum was comparable to controls. No clinically observable signs of toxicity were detected for offspring from all treatment groups.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
Under the test condition, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity for either sex can be established at 5000 ppm (298.8 mg/kg bw/day).The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 5000 ppm (298.8 mg/kg bw/day).
Executive summary:

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test conducted according to OECD Guideline 422 and in compliance with GLP, Menthanyl acetate multiconstituent was administered by dietary admixture (initially mixed with 2 % corn oil to avoid evaporation) to three groups of Sprague-Dawley Crl: CD® BR strain rats, for up to 63 consecutive days (including a three week maturation phase, pairing, gestation and early lactation for females), at dietary concentrations of 0, 800, 1500 and 5000 ppm (equivalent to a mean achieved dosage of 0, 50.7, 93.7 and 298.8 mg/kg bw/day respectively). Each dose group was subdivided into two phases: main phase (at 800 and 1500 ppm: 10 rats/sex/dose; at 0 and 5000 ppm: 5 males and 10 females/dose) and toxicity phase (5 female/dose). A control group was treated with basal laboratory diet (with 2% corn oil). Two recovery groups (5 rats/sex/dose) were treated with 5000 ppm or basal laboratory diet alone for 42 consecutive days and then maintained without treatment for a further 14 days. During the study, data was recorded on mortality, clinical signs, behavioural assessments, body weight change, food and water consumption, haematology, blood chemistry, mating performance, fertility and gestation length. All animals were subjected to a gross necropsy examination, selected organs were weighed and histopathological evaluation of selected tissues was performed. The clinical condition of offspring, litter size and survival, sex ratio and offspring bodyweight were assessed and macroscopic pathology investigations were undertaken.

No unscheduled deaths or treatment-related clinical signs were noted. No treatment-related effects were noted on behavioural, sensory reactivity and functional performance parameters. A significant reduction (-65 %) in body weight gain was evident in females treated with 5000 ppm during the first week of treatment. Food consumption and food efficiency were also adversely affected in these females during this period. The reduced food consumption was considered to reflect an initial reluctance to eat the diet admixture due to its low palatability. Recovery was evident thereafter. No treatment-related significant changes were detected after haematology and blood chemistry investigations. No treatment-related effects were detected in mating performance, fertility and gestation lengths. Main phase males from all treatment groups showed an increase in kidney and liver weight both absolute and relative to terminal body weight. A true dose related response however was not evident. Recovery males treated with 5000 ppm also showed an increase in absolute and relative liver weight. Toxicity phase females treated with 5000 ppm showed an increase in liver weight both absolute and relative to terminal body weight. Post mortem examinations did not reveal any treatment-related macroscopic findings for interim death or terminal kill offspring. Histopathology revealed microscopic abnormalities in liver (minimal to slight diffuse hepatocellular hypertrophy in main phase males and toxicity phase females at 1500 and 3000 ppm) and thyroid (minimal diffuse follicular cell hypertrophy in main phase males and toxicity phase females at 5000 ppm). These findings were fully reversible in recovery animals and considered either to be adaptive in nature or incidental and therefore not to represent an adverse effect of treatment. At 5000 ppm, microscopic changes in kidney (slight focal and unilateral tubular degeneration/regeneration, and minimal focal to multifocal and unilateral to bilateral corticomedullary mineralization) were observed in in main phase males, toxicity phase females and recovery animals (partly reversible). These findings were considered to be reflecting a minimal variant of alpha 2u-globulin nephropathy (males only) or premature stages of chronic progressive nephropathy which are not indicative of a hazard to human health. No treatment-related significant effects were noted on offspring litter size, sex ratio, viability, growth and development.

Under the test condition, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity when effects relevant to man are taken into account can be established at 5000 ppm (298.8 mg/kg bw/day).The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 5000 ppm (298.8 mg/kg bw/day).