Registration Dossier

Administrative data

Description of key information

Rat oral 2 yr NOAEL - 0.1 % in diet (non-guideline study); rat oral 16 wk NOAEL < 1 % (non-guideline study); rat oral 1 yr NOAEL ≥ 0.25 % (non-guideline study); rat oral 90 day NOAEL - 0.1 % in diet (non-guideline study).

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
Remarks:
combined repeated dose and carcinogenicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
No data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Meets generally accepted scientific standards, well documented and acceptable for assessment
Principles of method if other than guideline:
Male and female rats were fed test substance in their diet for 2 years and observed.
GLP compliance:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: A. Tuck & Son, Battlesbridge, Essex, United Kingdom
- Age at study initiation: No data
- Weight at study initiation: males mean weight 81 g; females mean weight 77 g.
- Fasting period before study: No data
- Housing: Grid floor metal cages, 4 animals per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: No data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 °C ± 2 °C
- Humidity (%): No data
- Air changes (per hr): Not quantified, but described as "ventilated".
- Photoperiod (hrs dark / hrs light): No data
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): No data
- Mixing appropriate amounts with: Reground Spratt's Laboratory Animal Diet No. 1
- Storage temperature of food: No data
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No data
Duration of treatment / exposure:
104 wks
Frequency of treatment:
Continuous - test substance included in ad libitum diet.
Remarks:
Doses / Concentrations:
0.02 %
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0.01 %
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0.5 %
Basis:
nominal in diet
No. of animals per sex per dose:
48 per sex per dose.
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: Based on previously published academic papers.
Positive control:
None
Observations and examinations performed and frequency:
OBSERVATIONS
- Extent of observations not specified
- Time schedule: Daily
- Rats showing signs of ill health were isolated, to be returned to their group if their condition improved, but otherwise to be killed and autopsied.

BODY WEIGHT
- Time schedule for examinations: Observations at 0 wks, 4 wks, 9 wks, 12 wks, 20 wks, 27 wks, 40 wks, 53 wks, 67 wks, 79 wks, 91 wks, 103 wks.

FOOD CONSUMPTION AND COMPOUND INTAKE
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY
- Body weight gain in kg/food consumption in kg per unit time × 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY
- Time schedule for collection of blood: Observations at 16 wks, 24 wks, 54 wks, 2 yrs
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 10 per sex (control, 0.1 %, and 0.5 % groups only)
- Parameters examined:
-- Haemoglobin, packed cell volume, erythrocyte count, total leucocyte count (control, 0.1 %, and 0.5 % groups)
-- Reticulocyte, differential white counts (control and 0.5 % groups only)
-- Blood serum analysed for urea, glucose, total albumin and activities of glutamic-pyruvic transaminase and lactic dehydrogenase (female control, female 0.5 %, male control, male 0.5% , male 0.02% groups at 2 yr only)

URINALYSIS
- Time schedule for collection of urine: 12 wks, 24 wks, 54 wks, 79 wks
- Collection
-- 6 hr period of water deprivation.
-- 2 hr period following water load of 25 mg/kg bw.
-- 4 hr period following water deprivation of 16 hr.
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- No. of animals: 10 per sex per dose (12 wks, 24 wks); 5 per sex for doses of 0, 0.1 %, 0.5 % (54 wks, 79 wks).
- Parameters examined: specific gravity, volume.
-- 6 hr samples examined semi-quantitatively for protein, glucose, ketone bodies, bile salts, blood.
-- 2 hr samples examined for No. of cells

NEUROBEHAVIOURAL EXAMINATION
- Time schedule for examinations: 2 wks, 15 wks, 21 wks, 62 wks, 84 wks
- Dose groups that were examined: 15 male control group, 15 male 0.5 % group, 15 female control group, 15 female 0.5 % group.
- Battery of functions tested: motor activity
- Details: Tested for ability to remain on rotarod at 11 rpm for 3 mins. No. of attempts required recorded, maximum attempts allowed 10.
Sacrifice and pathology:
GROSS PATHOLOGY: No data

HISTOPATHOLOGY
- Survivors at wk 104
- Macroscopic abnormalities noted.
- Weighing of brain, heart, liver, spleen, kidneys, stomach, small intestine, caecum (with and without contents), adrenal glands, gonads, pituitary and thyroid.
- Samples of brain, heart, liver, spleen, kidneys, stomach, small intestine, caecum, adrenal glands, gonads, pituitary, thyroid, salivary gland, trachea, lungs, aorta, thymus, lymph nodes, urinary bladder, colon, rectum, pancreas, uterus, mammary gland, prostate, seminal vesicles, skeletal muscle, eye, Harderian gland, spinal cord, sciatic nerve, brachial nerve and any other tissues of abnormal appearance fixed, in a 10 % buffered formalin. Microscopic examinations of these carried out on 5 µm paraffin wax sections stained with haematoxylin and eosin.
Statistics:
No data
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
not examined
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
- No. of deaths amongst 0.5 % group was less than control group, but not statistically significant.

BODY WEIGHT AND WEIGHT GAIN
- Body weights were significantly lower in females fed 0.5 % test material, although differences were small (less than 10 % of the control).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
- Food intake was slightly lower in females fed 0.5 % test material. In this group, there was no difference in water intake.

HAEMATOLOGY
- Statistical difference (P < 0.05) between treated and control rats - a high leucocyte count in wk 54 in males fed 0.5 % test substance. There was no parallel finding in the females. At wk 104, haematological values were similar in all groups.

URINALYSIS
- No consistent effects
- Semi-quantitative tests for glucose, bile salts, blood and ketones negative. Protein and microscopic constituents similar in control and test groups.

NEUROBEHAVIOUR
- No evidence of neuropathy

ORGAN WEIGHTS
- Isolated values for absolute or relative organ weights in the treated animals differed from the control groups, but none in the 0.5 % treatment.

HISTOPATHOLOGY: NON-NEOPLASTIC
- In general, the frequency of the histological findings was similar in the control and treated groups but male animals fed 0.5 % showed a greater incidence of fatty vacuolation of the liver.
- In males fed 0.1 % (but not 0.5 %), there were significantly more animals with liver necrosis. However, the extent of the lesions in affected rats was similar in treated and control animals, and there was no statistically significant difference in females.
- Dilation of the pancreatic duct occurred with great frequency in males fed 0.5 % test substance, and in females fed 0.02 %.
- A marginal treatment-related increase in dilation of lymph node sinusoids occurred in male but not female rats, the difference being statistically significant in rats fed 0.5 % test substance.

HISTOPATHOLOGY: NEOPLASTIC
- See table 9 in the attached supporting materials

Most of the tumours were encountered as isolated findings or in very low incidence in any one group with no statistically-valid differences between the treated and control groups. The exceptions to this were interstitial cell tumours of the testis, which occurred more frequently in the males given 0.02 % or 0.5 % test material, and pituitary adenomata, which were more common amongst females of the same groups that in the controls. In both cases, the incidence in the intermediate group did not differ significantly from the control group.

A number of malignant tumours of vascular origin were seen. Two haemangiosarcomata were present in female animals, one in the 0.1 % group and the other in the 0.5 % group. In males, similar tumours were found in single animals from the control and two lowest dose groups. In addition, haemangioendotheliomata were identified in one female in the 0.5 % group (in the liver) and in one in the 0.02 % group (in the uterus). Benign tumours of vascular origin (haemangiomata) were confined to one control male and 0.1 % dose female.

Lymphosarcomata were encountered in occasional animals in most groups. Affected were the spleen of a control male and multiple organs, including the lymph nodes, of a single male in each of the groups fed 0.02 % and 0.1 % test material. In females, lymphosarcomata were identified in the thyrus of two rats from the 0.02 % dose group and in the bone of one in the 0.5 % dose group. Two male animals, one from 0.02 % dose and one from the 0.1 % dose group had reticulum cell sarcomata.

Some malignant tumours of common origin were found in more than one treated animal. An osteosarcoma occurred in one male from the 0.02 % group and in one from the 0.5 % group, the tumour in the latter case being identified from a metastatic deposit in the lung. Spindle cell sarcoma was found in the large intestine of one male fed 0.5 % test material and in the bladder of a male fed 0.02 % and adrenal-gland cortical carcinomata occurred in 2 males (in the 0.02 % and 0.5 % groups). In females, mammary adenocarcinoma was found in 1 animal from each of the 2 higher dosage groups.

Single occurrences of malignant tumours in treated animals without a similar control finding included a uterine endometrial sarcoma and neurofibrosarcoma of the ovary in animals fed 0.5 % and 0.1 % test material respectively, while an astrocytoma was present in the brain of a male animal fed 0.1 % test material. An anaplastic carcinoma of the pancreas and basal-cell carcinoma of the skin were found in males from the high dose group.

There were more pituitary adenomata in the treated female rats than in the controls but Dunnington et al. considered such tumours to be particularly common in old rats and cite incidences of 50-70 % that have frequently been reported. They also reported that similar incidences 50-70 % have been reported in their laboratory in control animals. They thus considered the number seen in the treated animals as not unusual, and the difference in incidence between control and treated groups seems to have been due to a low incidence (42 %) in the control group rather than to treatment with the test material.

There were no statistically significant differences in the overall incidence of tumours, either benign or malignant, between the treated and control male rats. In the females there was a higher incidence of benign tumours, but this was no longer the case when the pituitary tumours were excluded and incidences of pituitary tumours do not seem to have been due to tumours.

Interstitial cell tumour of the testis was the only other individual tumour that occurred in treated rats with a frequency of statistical significance. Dunnington cite reports that such tumours are the most common testicular neoplasmin the rat. Control data from Dunnington et al.'s laboratory indicate an incidence in some studies as high as 10 % in Wistar rats, with much variation between groups. In this study there was an incidence of 5 % in the control animals, while in those fed 0.02 % test material there was an incidence of 23 % and in the group fed 0.5 % test material an incidence of 22 %. The incidence among the rats fed 0.1 % test material however was 3 % so there was no dose-response relationship.

All of the tumours were present in animals killed at the end of the study; none was found at earlier stages. The lack of any relationship between dosage and the number of tumours found or of any evidence for a shortening of the latent period suggested to Dunnington et al. that the distribution of these testicular tumours was more likely to be a reflection of variations in the normal incidence in rats than an effect of the test material, although the latter possibility cannot be ruled out entirely.

There was no dose-association pattern in the occurrence of other benign tumours in treated rats and all the types encountered were well documented in previous literature.

A number of malignant tumours occurred in treated animals but not in controls. However, reticulum-cell sarcoma, haemangiosarcoma, adrenal carcinoma, basel-cell carcinoma, endometrial sarcoma, spindle-cell sarcoma of the bladder and of the large intestine, an mammary adenocarcinoma have all been reported to arise spontaneously in the rat. Two of these types of tumour of particular interest to Dunnington et al: Lymphosacroma and reticulum cell sarcoma form a group of tumours that occur spontaneously in the lymphoretuclar system of ageing rats, and cite an incidence of 20 % that had been reported in the Furth strain of Wistar rat. In Dunnington's laboratory, there was an overall incidence of 3.3 % for males and 1.2 % for females among control Wistar animals. The overall incidences in the various groups were similar to this background figure and there was no evidence of any dose-relationship, particularly since none of these sarcomata were present in animals on the highest dose level. It seems likely therefore to Dunnington et al. that the incidence of lymphoreticular tumours recorded in the investigation was affected by the administration of the test material.

Vascular neoplasms formed the second group of tumours that occurred with relatively high frequency in the study. Malignancies of vascular origin were present in the lymph nodes, liver, kidney and uterus.

The overall incidence of tumours of vascular origin classified as malignant (haemangiosarcoma and haemangioendothelioma) was 1, 1, 1,0 in the control, 0.02 %, 0.1 % and 0.5 % dose males respectively, and 0, 1, 1, 2 in the females. These incidences were not statistically-significant and showed no overall dose relationship. Although these tumours were classified as malignant, doubt remains as to their exact nature because of a combination of local invasive properties and benign cytological features. This problem had been encountered with vascular tumours at other sites.

Although some of the benign tumours occurred with increased frequency in the treated animals, Dunnington et al. found that there was no conclusive evidence to link their development with treatment with the test material.
Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects
Dose descriptor:
NOAEL
Effect level:
ca. 35 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: overall effects
Dose descriptor:
NOAEL
Effect level:
ca. 60 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: overall effects
Critical effects observed:
not specified
Conclusions:
Dunnington et al. found that the no observed effect level of the test material to be 0.1% in the diet of Wistar Rats. This is equivalent to 35mg/kg bw/d for Males and 60mg/kg bw/d for Females.
Endpoint conclusion
Dose descriptor:
NOAEL
35 mg/kg bw/day
Study duration:
chronic
Species:
rat

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

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-02-24 to 2013-01-30
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study to reproduction/developmental screening guideline via dermal route which is sufficiently similar to subacute repeat dermal study.
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. QA statement)
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxfordshire, United Kingdom
- Age at study initiation: Approximately 11 weeks old
- Weight at study initiation: Males 282 g to 337 g; females 176 g to 224 g
- Housing: Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. During the pairing phase, the animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis. Following evidence of successful mating, the males were returned to their original cages. 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): A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK) was available ad libitum.
- Water (e.g. ad libitum): Mains drinking water was supplied from polycarbonate bottles attached to the cage ad libitum.
- Acclimation period: The animals were acclimatised for thirteen days during which time their health status was assessed. During this acclimatisation period preparation of the test site (i.e. clipping) and bandage training for five consecutive days was performed. On the initial day of bandage training, the bandages were only applied for approximately 3 hours, for the remaining days bandage training matched the exposure period applied when animals were being treated.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ºC ± 3 ºC
- Humidity: 50 % ± 20 % Relative humidity exceeded this target range on two transient occasions (achieved range 45-72 %RH) but these deviations were considered to have had no impact on the scientific validity or integrity of the study.
- Air changes: ≥ 15 per hour
- Photoperiod: 12 hrs dark / 12 hrs light
Vehicle:
other: 2% Carboxymethylcellulose/1% Tween 80
Details on exposure:
TEST SITE
- Area of exposure: the dorso-lumbar region
- % coverage: approximately 10% of the total body surface area
- Type of wrap if used: The site of application was semioccluded
using a piece of porous gauze covered with a self-adherent bandage.

REMOVAL OF TEST SUBSTANCE
- Washing: The test item formulation removed from the dosing test site and the exposed area was decontaminated with 0.9% saline.
- Time after start of exposure: Following six hours of exposure.

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): The dose volume employed was 3 ml/kg
- Concentration (if solution): The volume of test and control item administered to each animal was based on the most
recent scheduled body weight and was adjusted at regular intervals to ensure the dosage for each dosage group was maintained; 0, 33.33, 100, 333.33 mg/mL.
- Constant volume or concentration used: yes
- For solids, paste formed: Not applicable

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Amount(s) applied (volume or weight with unit): The dose volume employed was 3 ml/kg
- Concentration (if solution): No data

USE OF RESTRAINERS FOR PREVENTING INGESTION: No
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability and homogeneity of the test item formulations were determined by Harlan Laboratories Ltd, Shardlow, UK, Analytical Services as part of this study. Results show the formulations to be stable for at least 18 days. Formulations were therefore prepared fortnightly and stored at approximately 4 ºC in the dark.
Duration of treatment / exposure:
The test item formulation was applied daily to the exposed region by a plastic syringe for 51 consecutive days for males, and up to Day 19 of gestation for females.
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
100 mg/kg bw/day
Basis:
nominal per unit body weight
Remarks:
Doses / Concentrations:
300 mg/kg bw/day
Basis:
nominal per unit body weight
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:
nominal per unit body weight
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
STUDY SCHEDULE
- Groups of 10 male and 10 female animals were dosed according to dose group for 14 days prior to pairing.
- On Day 15, following decontamination of the exposure site, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
- Animals were returned to their original holding cages during the exposure period.
- Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
- Treatment was continued for males until study termination. Pregnant females were treated up to and including Day 19 of gestation.
- Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Litter size, offspring weight and sex, surface righting and clinical signs were also recorded during this period.
- The male dose groups were killed and examined macroscopically on Day 52.
- The termination of the males was delayed (and dosing extended) to allow for the results of mating for the majority of the females to be known prior to male necropsy.
Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS
All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before each application, one hour after application and after removal of the porous gauze and decontamination of the test site. All observations were recorded.

LOCAL IRRITATION
Prior to each application of the test item, the dose test site was examined for any signs of irritation. Any irritation observed was scored according to the scheme described by Draize J H (1959).

BODY WEIGHT
- Time schedule for examinations: Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until mating was evident. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.

FOOD CONSUMPTION AND COMPOUND INTAKE
During the pre-pairing period, weekly food consumption was recorded for each cage of adults until pairing. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded during the lactation period (Days 1-4).

Weekly food efficiency (body weight gain/food intake) was calculated retrospectively for males and for females during the pre-pairing phase. Due to offspring growth and milk production for lactation, food efficiency for females could not be accurately calculated for females during gestation and lactation.

WATER CONSUMPTION AND COMPOUND INTAKE
- Time schedule for examinations: Water intake was observed daily by visual inspection of water bottles for any overt changes.
- Intergroup differences did not indicate any need for more formal gravimetric measurements.
Sacrifice and pathology:
SACRIFICE
Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 52. Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 25 post coitum.

PATHOLOGY
For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5 % ammonium polysulphide solution (Salewski 1964). For pregnant females the number of corpora lutea in the ovaries was also recorded.

All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

ORGAN WEIGHTS
The epididymides and testes were removed from terminal kill adult males dissected free from fat and weighed before fixation.

HISTOPATHOLOGY
Samples of the following tissues were preserved from all animals from each dose group, in buffered 10 % formalin: Coagulating gland (males only), prostate (males only), seminal vesicles (males only), gross lesions, ovaries (females only), treated/untreated skin, mammary gland (females only), uterus/cervix (females only), pituitary, Vagina (females only).

Samples of the following tissues were preserved in Bouin’s fluid then transferred to 70 % Industrial Methylated Spirits (IMS) approximately 48 hrs later: Testes (males only), epididymides (males only).

The tissues from control and 1000 mg/kg bw/day dose group animals and any animals which failed to mate or did not achieve a pregnancy were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with Haematoxylin and Eosin for subsequent microscopic examination. In addition, sections of testes and epididymides from all control and 1000 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined. Detailed qualitative examination of the testes was performed taking into account the tubular stages of spermatogenic cycle.
Statistics:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:
Body Weight, Body Weight Change, Food Consumption during gestation and lactation,
Pre-Coital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Corpora Lutea, Implantation Sites, Implantation Losses, Viability Indices, Offspring Body Weight, Offspring Body Weight Change, Offspring Surface Righting, Absolute Organ Weights, Body Weight-Relative Organ Weights.
Data were analysed using the decision tree from the Provantis Tables and Statistics Module as detailed below:
Where appropriate, data transformations were performed using the most suitable method

The homogeneity of variance from mean values was analysed using Bartlett's test. Intergroup variance were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any 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 shows non-homogeneity of means, the data were analysed by a stepwise Dunnett's (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (nonparametric). Data not analysed by the Provantis data capture system were assessed separately using the SPSS statistical package. Initially, the homogeneity of the data was assessed using Levene's test.
Clinical signs:
no effects observed
Dermal irritation:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
MORTALITY
There were no unscheduled deaths on the study.

CLINICAL OBSERVATIONS
Clinical signs observed on the study were minimal and were unrelated to treatment.

BODY WEIGHT
Body weight gain was unaffected by treatment for both sexes throughout the study, which included for females, gestation and lactation phases, at 100, 300 and 1000 mg/kg bw/day.
At 300 and 1000 mg/kg bw/day higher bodyweight gain for males, attained statistical significance compared to control during Days 15 to 22 but there was no dosage relationship. These differences were considered to reflect normal biological variation and were of no toxicological significance.

FOOD CONSUMPTION AND FOOD EFFICIENCY
Food consumption and food conversion efficiency was considered to have been unaffected by treatment for both sexes throughout the study, which included for females, gestation and lactation phases, at 100, 300 and 1000 mg/kg bw/day. At 300 and 1000 mg/kg bw/day food intake was higher than control during the final week of gestation. Although statistical significance was reached at the highest dosage, these differences were considered to reflect normal biological variation rather than an effect of treatment. Additionally food consumption during lactation was lower than control at all dosages during lactation; differences failed to attain statistical significance and there was no dosage relationship, The lower food consumption was considered to reflect particularly high food intake for two control females and was unrelated to treatment.

REPRODUCTIVE PERFORMANCE: MATING
There was no adverse effect of treatment on mating performance at 100, 300 and 1000 mg/kg bw/day.
It was noted that some treated animals show a longer pre-coital interval than their control counterparts; the incidence and distribution of these animals did not indicate any dosage relationship and this finding was considered to be incidental and unrelated to treatment.
For the majority of animals observed evidence of mating was good; one control female (No. 18) and one at 100 mg/kg day (No. 36) showed poor evidence of mating but only the control animal failed to achieve pregnancy.

REPRODUCTIVE PERFORMANCE: FERTILITY
There was no adverse effect of treatment on fertility at 100, 300 and 1000 mg/kg bw/day. At both 300 and 1000 mg/kg bw/day two females failed to achieve pregnancy, however three of the control females also failed to achieve pregnancy. This slightly lower than anticipated pregnancy rate may reflect the daily separation of the males and females during the two week pairing period as part of dermal dose administration procedure.

GESTATION LENGTH
No treatment-related effects were detected in the length of gestation between control and treated groups, with all littering females showing a gestation length between 22½ and 24 days.

NECROPSY
Adult macroscopic necropsy findings were restricted to small and flaccid testes and small epididymides for one male at 1000 mg/kg bw/day. This animal failed to induce pregnancy in its female partner. In isolation this finding was considered to be incidental and unrelated to treatment.

ORGAN WEIGHTS
Mean absolute and body weight relative testis and epididymis organ weights were unaffected by treatment at 100, 300 and 1000 mg/kg bw/day.

HISTOPATHOLOGY
Examination of the skin (test site) and reproductive organs for both sexes did not indicate any adverse effect of treatment.
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects
Critical effects observed:
not specified

Dermal administration of Ethyl-3-methyl-3-phenyloxirane-2-carboxylate at dosages up to 1000 mg/kg bw/day was well tolerated by the adult animals with no adverse effects on survival, clinical condition, body weight gain, food consumption or macroscopic necropsy findings and subsequent microscopic evaluation. Within the context of this study, the No Observed Effect Level (NOEL) for adult toxicity was 1000 mg/kg bw/day.

Conclusions:
A study was conducted to OECD 421 (reproduction/development screening test), via the dermal route, which exposed rats to strawberry pure at doses up to at doses up to 1000 mg/kg bw/d. Male rats were exposed for 52 days and female rats for approx. 33 days including a 14 day pregnancy period. The adult NOAEL was found to be 1000 mg/kg bw/d.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Oral route

Mason et al. (1978) found, in a 90 day subchronic oral toxicity study on rats, that the NOAEL for the test material was 0.1 % in diet. This corresponded to a dose of 150 mg/kg bw/d and 60 mg/kg bw/d at the beginning and end of the study, respectively. Adverse effects were found in rats treated with 0.5 % in diet.

Dunnington et al. (1981) found, in a 2 yr chronic oral toxicity study also on rats, that the NOAEL for the test material was 0.1 % in diet. They report this corresponded to doses of 35 mg/kg body weight/day in males and 60 mg/kg body weight/day in females.

Previously, Hagan et al. (1967) had reported two studies. In a 16 wk study, the only dose of 1 % in diet produced adverse effects. In their 1 yr chronic oral toxicity study, the only dose of 0.25 % in diet produced no adverse effect.

A 28 day repeat dose study cannot be justified and is not required because of the availability of longer-term studies.

Analysis by Mason et al. (1978) of commercially available strawberry aldehydes by gas-liquid chromatography identified variability in specifications of those substances, and Mason et al. criticised previous studies that lacked detail on the substance specifications. The studies by Hagan et al. are also somewhat old and the methodology does not appear to be to modern standards. Taking this into consideration, the studies by Hagan et al. (1967) have not been considered particularly reliable. The 16 wk study was not very thorough although its finding that a dose of 1 % in diet produced adverse effects is consistent with Mason et al. (1978). However, the NOAEL figure in the 1 yr study by Hagan et al. (1967), 2500 ppm or 0.25 % in diet, is higher than that obtained by either Mason et al. (1978), or Dunnington et al. (1981). This may have been because the study lacked rigour and therefore failed to detect adverse effects. The value used for the chemical safety assessment will be based on those obtained in the later studies. This also satisfies the principal that the effect levels should be identified in a conservative manner to avoid overestimating them.

According to Annex VIII section 8.6.1 of Regulation (EC) No. 1907/2006, "the short-term toxicity study (28 days) does not need to be conducted if...a reliable sub-chronic (90 days) or chronic toxicity study is available, provided that an appropriate species, dosage, solvent and route of administration were used". Such studies are available and the 28 day study is an in vivo test, so conducting a 28 day study short-term repeated dose toxicity study is inappropriate.

Dermal route

Regarding dermal exposure, the substance is classified as a skin sensitiser. This classification should apply to all mixtures containing the substance at > 1.0 % w/w, per Table 3.4.3 of Regulation (EC) No. 1272/2008. Appropriate risk management measures should be implemented to avoid dermal exposure to the substance at or above this level. Skin contact in production and/or use is therefore unlikely. Any one-off accidents would represent acute exposure to the substance, not subchronic or chronic repeat doses. Repeat exposure via the dermal route should therefore be ruled out at these concentrations, although may still occur at lower concentrations. We do not consider dermal exposure to be the most likely route of human exposure.

Data are however presented from a study to OECD guideline 421 (reproduction/developmental toxicity screening test). In this, rats were dermally exposed to the test material at doses up to 1000 mg/kg bw/d for extended periods. Male rats were exposed for 52 days; females for approx. 33 days. The NOAEL from this study was 1000 mg/kg bw/d.

Further dermal testing is therefore not proposed.

Inhalation route

The vapour pressure of the substance is 0.23 Pa at 20 °C. This is considered low and therefore human exposure via inhalation is also unlikely. Further inhalation testing is therefore not proposed.

Justification for classification or non-classification