Tricyclodecanedimethanol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

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:

16 November 2009 - 08 January 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA, Health Effects Test Guidelines OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000.

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:WI(Han).

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories France, L'Arbresle Cedex, France.
- Age at study initiation: Approximately 11 weeks.
At the start of treatment the animals were 11 weeks instead of 10 weeks old. A slight deviation in age does not affect the study integrity. Mating started shortly after the animals had attained full sexual maturity according to the OECD 422 guideline.
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages (MIII type, height 18 cm).
Lactation: Pups were kept with the dam until termination in Macrolon cages (MIII type, height 18 cm).
General: Sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. Certificates of analysis were examined and then retained in the NOTOX archives. During activity monitoring, animals were housed individually in Macrolon cages (MIII type; height 15 cm) with sterilised sawdust as bedding material. No cage-enrichment was provided during activity monitoring.

- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: At least 5 days prior to start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.8 – 21.4°C
- Humidity (%): 32 - 94%
Temporary deviations from the minimum level of temperature and from the minimum and maximum level relative humidity occurred in the animal room. Laboratory historical data do not indicate an effect of the deviations.

- Air changes (per hr): approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial light and 12 hours darkness per day. Temporary fluctuations from the light/dark cycle (with a maximum of 1 hour) occurred due to performance of pupillary reflex tests in the room. Based on laboratory historical data, these fluctuations were considered not to have affected the study integrity.

IN-LIFE DATES: From: 16 November 2009 To: 08 January 2010.

Route of administration:

oral: gavage

Vehicle:

other: 20% (w/w) Ethylacetate (density 0.902 g/mL) in propylene glycol (density 1.036 g/mL).

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenised to a visually acceptable level. The test substance was suspended in ethylacetate to form a homogenous suspension. Subsequently, propylene glycol was added under constant stirring. Adjustment was made for the density of the vehicle (ethylacetate and propylene glycol) and for the test substance (1.107 g/cm3).

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at NOTOX.
- Concentration in vehicle: 30, 70 and 120 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: A maximum of 13 instead of 14 days was allowed for mating. All females, except one group 3 female, had mated in this period. It was considered that one additional day of mating would not have resulted in successful mating of that non-mated female. A sufficient number of pregnant females per dose group was available to make an adequate assessment of the study data.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage, by staging of the oestrus cycle and/or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): Females were individually housed in Macrolon cages (MIII type, height 18 cm).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted during the treatment phase, according to a validated method (NOTOX project 491999). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).

The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

RESULTS:
The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).

The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%) and the entire range of formulations were stable when stored at room temperature for at least 6 hours.

Duration of treatment / exposure:

Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 42-53 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation. Female no. 60 (group 2) and no. 78 (group 4) were not dosed during littering.

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to scheduled necropsy.

Details on study schedule:

- Age at mating of the mated animals in the study: Approximately 13 weeks

Remarks:

Doses / Concentrations:
0, 150, 350 and 600 mg/kg/day
Basis:

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on results of the dose range finding study (NOTOX Project 492082). See attachment.
Results of the dose range finding study are described in End point study record: Repeated dose toxicity: oral. notox 491996.
- 5 animals/sex/group were randomly selected at allocation for functional observations, clinical pathology, macroscopic examination (full list), organ weights (full list) and histopathology:
Males: the first 5 males per group
Females: with live offspring only

Positive control:

no

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily (early morning/late afternoon)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least immediately after dosing, detailed clinical observations were made in all animals. Once prior to start of treatment and at weekly intervals this was also performed outside the home cage in a standard arena. Arena observations were not performed when the animals were mating, or housed individually.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on Days 1 and 4.
For one female of Group 4 no body weight was determined during the post-coitum period as mating of this female was overlooked. Body weights were determined during the mating period and sufficient data was available to make a thorough assessment.

FOOD CONSUMPTION: Yes
- Weekly, for males and females. Food consumption was not recorded during the mating period. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.
For one female of Group 4 no food consumption was determined during the post-coitum period as mating of this female was overlooked. Sufficient food consumption data is available to make a thorough assessment.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m.
- Anaesthetic used for blood collection: Yes iso-flurane
- Animals fasted: yes, but water was available
- How many animals: 5 males/group (females: with live offspring only)
- Parameters examined were: white blood cells, differential leucocyte count , neutrophils, lymphocytes, monocytes, eosinophils, basophils, red blood cells, reticulocytes, red blood cell distribution width, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelets, prothrombin time, activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m.
- Animals fasted: yes, but water available
- How many animals: 5 males/group (females: with live offspring only)
- Parameters examined were: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total protein, albumin, total bilirubin, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate, bile acids.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested during lactation (all before blood sampling).
- Dose groups that were examined: all
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, grip strength and motor activity

Oestrous cyclicity (parental animals):

not determined

Sperm parameters (parental animals):

Parameters examined in all male parental animals:
testis weight, epididymis weight.
in addition, for 5 males of the control and high dose group, slides of the testes were prepared to examine staging of spermatogenesis.

Litter observations:

PARAMETERS EXAMINED:
Each litter was examined to determine the following, if practically possible:

Mortality / Viability:
The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.

Clinical signs:
At least once daily, detailed clinical observations were made in all animals.

Body weights:
Live pups were weighed on Days 1 and 4 of lactation.

Sex:
Sex was determined for all pups on Days 1 and 4 of lactation (by assessment of the ano-genital distance).

GROSS EXAMINATION OF DEAD PUPS: Yes
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated. Pups from female no. 51 that died spontaneously were preserved in 10% buffered formalin for possible further examination.

Postmortem examinations (parental animals):

SACRIFICE
All animals were fasted overnight (with a maximum of approximately 22 hours) prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy were deeply anaesthetised using iso-flurane vapor (Abbott Laboratories Ltd., Hoofddorp, The Netherlands) and subsequently exsanguinated. Necropsy was conducted on the following days:

- Male animals: Following completion of the mating period (a minimum of 28 days of dose administration).
- Maternal animals: All surviving animals on lactation Days 5-6.
- Female which failed to deliver: Post-coitum Day 29 (female with evidence of mating)
- Female which died spontaneously: As soon as possible after death, within 24 hours.

Several males were necropsied later than after a maximum of 20 hours fasting, i.e. with a maximum of approximately 2 hours. The fasting period was only slightly longer and was not considered to have adversely affected the clinical laboratory, macroscopic or microscopic findings.

One female which failed to deliver was necropsied on Day 29 Post-coitum instead of between Days 25-27. Female was not pregnant. An extended treatment duration was not considered to have adversely affected overall interpretation of the study results

GROSS NECROPSY
All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded. The number of former implantation sites and corpora lutea was recorded for all paired females.

Samples of the following tissues and organs were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands):

Selected 5 animals/sex/group and the female that died spontaneously#: Identification marks: not processed, Ovaries, Adrenal glands, (Pancreas), (Aorta), Peyer's patches (jejunum, ileum) if detectable, Brain (cerebellum, mid-brain, cortex), Pituitary gland, Caecum, Preputial gland, Cervix, Prostate gland, Clitoral gland, Rectum, Colon, (Salivary glands (mandibular, sublingual)), Duodenum, Sciatic nerve, Epididymides*, Seminal vesicles including coagulating gland, Eyes with optic nerve (if detectable) and Harderian gland*, Skeletal muscle, (Skin), Female mammary gland area, Spinal cord (cervical, midthoracic, lumbar), Femur including joint, Spleen, Heart, Sternum with bone marrow, Ileum, Stomach, Jejunum, Testes*, Kidneys, Thymus, (Larynx), Thyroid including parathyroid (if detectable), (Lacrimal gland, exorbital), (Tongue), Liver, Trachea, Lung infused with formalin, Urinary bladder, Lymph nodes (mandibular, mesenteric), Uterus, (Nasopharynx), Vagina, (Oesophagus), All gross lesions.

All remaining animals and the female which failed to deliver$: Cervix, Prostate gland, Clitoral gland, Seminal vesicles including coagulating gland, Epididymides*, Testes*, Ovaries, Uterus, Preputial gland, Vagina, Identification marks: not processed, All gross lesions.

# Recognizable fetuses were examined externally, sexed, euthanized by decapitation (if necessary) and preserved in 10% neutral-buffered formalin.
* Fixed in modified Davidson's solution (prepared at NOTOX using Formaldehyde 37-40%, Ethanol, Acetic acid (glacial)(all Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)) and transferred to formalin after fixation for at least 24 hours.
$ In case no macroscopically visible implantation sites were present, nongravid uterus was stained using the Salewski technique (Salewski 1964) in order to detect any former implantation sites (Salewski staining prepared at NOTOX using Ammoniumsulfide-solution 20% (Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)).

Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.

ORGAN WEIGHTS: Yes
The following organ weights and terminal body weight were recorded from the following animals on the scheduled day of necropsy:

Selected 5 animals/sex/group: Adrenal glands, Spleen, Brain, Testes, Epididymides, Thymus, Heart, Uterus (including cervix) , Kidneys, Prostate*, Liver, Seminal vesicles including coagulating glands*, Ovaries, Thyroid including parathyroid*.
* weighed when fixed for at least 24 hours.

All remaining males: Epididymides, Testes.

HISTOTECHNOLOGY: Yes
All organ and tissue samples, as defined under Histopathology (following), were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands).

Of the selected 5 males of the control and high dose group, additional slides of the testes were prepared to examine staging of spermatogenesis. The testes were processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin (Klinipath, Duiven, The Netherlands).

HISTOPATHOLOGY: Yes
The following slides were examined by a pathologist:
-The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4.
-The additional slides of the testes of the selected 5 males of Groups 1 and 4 to examine staging of spermatogenesis.
-The preserved organs and tissues of the female that died spontaneously.
-All gross lesions of all animals (all dose groups).
-The reproductive organs (cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testis, uterus, and vagina) of all animals that failed to mate, conceive, sire or deliver healthy pups:
Group 2: One male and one female (the female was found dead at littering)
Group 3: One male and one female (failed to conceive/sire)

Inadvertently, a few tissues were not available for histopathology. Reasons for missing a few tissues included that these tissues were not discernable at necropsy or trimming, or were erroneously not collected at necropsy. Missing tissues are listed in raw data and pathology report. Sufficient data was available for histopathological evaluation.

All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.

Postmortem examinations (offspring):

SACRIFICE
Pups surviving to planned termination were killed by decapitation on lactation Days 5 or 6.

GROSS NECROPSY
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated. Pups from female no. 51 that died spontaneously were preserved in 10% buffered formalin for possible further examination.

HISTOPATHOLOGY / ORGAN WEIGTHS
no

Statistics:

The following statistical methods were used to analyse the data:
-If the variables could be assumed to follow a normal distribution, the Dunnett-test (Dunnett, 1955) (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
-The Steel-test (Miller, 1981) (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
-The Fisher Exact-test (Fisher, 1950) was applied to frequency data.

The following additional methods of statistical analysis were used:
After testing for normality with the Shapiro-Wilk test, the number of corpora lutea was transformed by using x2 obtain a normal distribution. This was followed by an ANOVA. The Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control group.

An attempt was made to transform the number of implantation sites by using 1/x, log x, x2 and √x. However, a normal distribution was not obtained. Therefore, the number of implantation sites was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine inter-group differences, followed by the Wilcoxon test to compare the treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.

Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Reproductive indices:

For each group, the following calculations were performed:

Percentage mating females: Number of females mated/Number of females paired x 100

Fertility index females: Number of pregnant females/Number of females paired x 100

Conception rate: Number of pregnant females/Number of females mated x 100

Gestation index: Number of females bearing live pups/Number of pregnant females x 100

Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

Percentage live males at First Litter Check: Number of live male pups at First Litter Check/Number of live pups at First Litter Check x 100

Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100

Percentage of postnatal loss Days 0-4 of lactation: Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check x 100

Viability index: Number of live pups on Day 4 of lactation/Number of pups born alive x 100

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

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY:
No mortality occurred during the study period that was considered to be related to treatment with the test substance.

One female at 150 mg/kg/day died during delivery. No cause of death could be determined for this animal. Since no further mortality occurred among this dose group or in the other dose groups, this death was considered to be unrelated to treatment.

No clinical signs indicative of treatment-related toxicity were noted during the observation period.

Salivation observed among all animals at 350 and 600 mg/kg/day and at lower incidence at 150 mg/kg/day was considered to be a physiological response (eg. to taste of the formulation) rather than a sign of systemic toxicity, considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing).

Rales, and at lower incidence lethargy, shallow or laboured respiration, piloerection, uncoordinated movements and hunched posture were observed among the dose groups without a clear dose-related incidence. The occurrence of these signs was of an intermittent/transient nature (i.e. not related to the duration of treatment) and were therefore not considered to be toxicologically relevant.

A single female at 350 mg/kg was noted with a swelling of the left and right axillary regions for a few days during the post-coitum and lactation periods. Upon macroscopic examination this animal was found with a hard, gray-white nodule in the subcutis of the axillary region which was determined to be an adenocarcinoma of the mammary gland at histopathological examination. This was not considered to be treatment-related.

Other incidental findings consisted of a wound, scabbing and alopecia of various body parts. The incidence of these findings occurred within the background range of findings encountered for rats of this age and strain, which are housed and treated under the conditions of this study. No toxicological relevance was therefore ascribed to these observations.

BODY WEIGHT (PARENTAL ANIMALS)
No toxicologically relevant changes in body weights and body weight gain were noted.

The slightly lower mean body weights and body weight gain of males at 600 mg/kg/day during the premating and mating period (achieving a level of statistical significance on several occasions) remained within the range considered normal for rats of this age and strain, and thus were not toxicologically relevant.

Other statistically significant changes in body weight (gain) occurred in the absence of a dose- and time-related trend, and were of a very slight nature. These changes consisted of a lower body weight of females at 350 mg/kg/day on Day 1 of lactation, and a higher body weight gain of females and 150 and 600 mg/kg/day on Day 4 of the Post-coitum phase. No toxicological relevance was ascribed to these changes.

One female at 350 mg/kg/day showed a notable weight loss on Day 7 of the Post-coitum phase as (14%). As the incidence of this finding was unrelated to the dose, this was considered to be without toxicological relevance.

FOOD CONSUMPTION (PARENTAL ANIMALS)
No treatment-related changes in food consumption before or after allowance for body weight were noted.

The statistically significant higher absolute and relative food consumption of females at 600 mg/kg/day over Days 0-4 of the Post-coitum phase was slight in nature and was absent during the remainder of the Post-coitum and Lactation period. No toxicological relevance was ascribed to these changes.

Two females at 350 mg/kg/day showed a severely reduced food intake during the Post-coitum phase on Days 7-11 and 4-7 respectively. As these were incidental occurrences, no toxicological relevance was ascribed to these changes.

HAEMATOLOGY (PARENTAL ANIMALS):
No toxicologically relevant changes occurred in haematological parameters of treated rats.

Any statistically significant changes in haematological parameters were considered to be of no toxicological relevance as these occurred in the absence of a dose-related trend and/or remained within the range considered normal for rats of this age and strain. These changes consisted of lower mean corpuscular haemoglobin concentrations (MCHC) for males at 150, 350 and 600 mg/kg/day, and higher reticulocyte counts and lower haemoglobin levels for females at 350 mg/kg/day.
Any notably high or low individual haematological values occurred in the absence of a dose-related incidence, and were considered to be of no toxicological relevance. These individual variations included higher relative neutrophil counts and lower lymphocyte counts in male no. 23 (Group 3) and female no. 53 (Group 2), and higher red cell distribution width (RDW) in male no. 35 (Group 4).

CLINICAL CHEMISTRY (PARENTAL ANIMALS):
The following (statistically significant) changes in clinical biochemistry parameters distinguished treated animals from control animals:
- Higher creatinine in males* and females at 600 mg/kg/day,
- Higher total bilirubin in females at 350* and 600* mg/kg/day,
- Higher urea in females at 600 mg/kg/day,
- Lower glucose levels in males at 150, 350 and 600 mg/kg/day.
* exceeding the range considered normal for rats of this age and strain.

The statistically significant higher sodium levels for males at 350 mg/kg and chloride levels for males at 150 and 600 mg/kg/day were considered to be of no toxicological relevance as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain.

These changes were generally slight in nature (i.e. within the normal range or slightly exceeding the range), and occurred in the absence of any corroborative morphological changes indicative of organ dysfunction. Therefore, the changes in clinical biochemistry parameters were not considered to be toxicologically relevant.

NEUROBEHAVIOUR (PARENTAL ANIMALS):
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals.
No toxicologically relevant changes in motor activity were observed.

Locomotor activity for females at 350 and 600 mg/kg/day appeared higher than controls. The higher counts for both the high and low sensors for these females were largely driven by one female in each of the two groups with notably high sensor counts (nos. 65 and 73, respectively). When the data were re-calculated excluding the sensor counts for these females, high sensor values were similar to control values, whilst low sensor values still appeared higher for females at 350 and 600 mg/kg/day, being statistically significant for females at 600 mg/kg/day. However, the means remained within the range considered normal for rats of this age and strain, and clinical signs did not support these variations. Therefore, no toxicological relevance was ascribed to these variations.


REPRODUCTIVE DATA (PARENTAL ANIMALS)
No treatment-related effects on reproductive parameters were noted.

The mating, fertility and conception indices, precoital time, number of corpora lutea and implantation sites were unaffected by treatment.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No toxicologically significant changes were noted in organ weights and organ to body weight ratios.

The statistically significant higher kidney to body weight ratio of males at 600 mg/kg/day was considered to be without toxicological relevance since the mean remained within the normal range for rats of this age and strain, no dose-related increase in absolute kidney weights was observed among male dose groups, and no histopathological correlates were found. The higher testes to body weight of males at 600 mg/kg/day occurred due to a lower terminal body weight since absolute weights were similar to control levels.

The statistically significant lower liver to body weight ratios of males and adrenal to body weight ratios of females at 150 mg/kg/day occurred in the absence of a dose-related trend, and were therefore considered to not be toxicologically relevant.

Other organ weights and organ to body weight ratios among the dose groups were similar to control levels.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Macroscopic observations at necropsy did not reveal any alterations that were considered to be toxicologically relevant.

One female at 350 mg/kg/day, showed a gray-white hard nodule of the left axillary region subcutis. This finding corresponded to a swelling of the axillary region noted during the in-life phase, and to an adenocarcinoma of the mammary gland which was not considered to be treatment related.
The female that died spontaneously at 150 mg/kg/day was noted with yellowish contents of the gastro-intestinal tract, several reddish foci on the thymus, red discolouration of the mandibular lymph nodes, and cloudiness of both eyes. No relationship with treatment was established for these findings.

Incidental findings included alopecia and/or scabbing of various body parts, reddish foci on the lungs, stomach and/or thymus, reddish or red-brown discoloration of the liver, thymus, reddish discoloration of the mesenteric lymph node, irregular surface of the forestomach, a red-brown or tan focus on the clitoral gland, and accentuated lobular pattern of the liver. The incidence of these findings was within the background range of findings that are encountered among rats of this age and strain, did not show a dose-related incidence trend. These necropsy findings were therefore considered to be of no toxicological significance.

No macroscopic abnormalities were noted among surviving females at 150 mg/kg/day.

HISTOPATHOLOGY (PARENTAL ANIMALS)
There were no treatment-related microscopic findings.

One female at 600 mg/kg had minimal forestomach inflammation, hyperplasia and hyperkeratosis and slight forestomach edema. In the absence of any other forestomach lesions in the animals at this dose level, and considering that this finding sometimes occurs in this type of study, it was not considered to be treatment-related.

One female at 350 mg/kg had a mammary gland adenocarcinoma which correlated to the gray-white nodule finding recorded upon macroscopic examination. Although mammary gland adenocarcinoma is a rare finding at this age, mammary gland lesions are one of the most commonly occurring natural neoplastic lesions in this rat strain. As such, this finding was regarded as not treatment-related.

All other microscopic findings recorded were considered to be within the normal range of background pathology encountered in Wistar-Han rats of this age and strain.

No abnormalities were seen in the reproductive organs of suspected non-fertile animals which could account for infertility and the assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis.

Dose descriptor:

NOAEL

Effect level:

600 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects, highest dose tested

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

DEVELOPMENTAL DATA
No toxicologically relevant effects on developmental parameters were observed.
The gestation index and duration of gestation were unaffected by treatment.

Parturition and maternal care
No deficiencies in maternal care were observed. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No signs of difficult or prolonged parturition were noted among the pregnant females, with the exception of a single female at 150 mg/kg that died during delivery. No cause of death could be established for this female.

Early postnatal pup development
The number of dead and living pups at first litter check, sex ratio, postnatal loss, viability index, and early postnatal pup development (mortality, clinical signs, body weight and external macroscopy) were unaffected by treatment.

Mortality
Three pups of the control group, one pup at 150 mg/kg/day, two pups at 350 mg/kg/day and one pup at 600 mg/kg/day were found dead or missing during the first days of lactation. No relationship with treatment was established for these deaths.
All pups of the female that died during delivery were found dead, and one early resorption was found.

Clinical signs
Incidental clinical symptoms of pups consisted of small size and pale appearance. No relationship with treatment was established for these observations and they were considered to be of no toxicological significance.

Body weight
There were no differences in body weights between control and treated pups at any dose level.

Macroscopy
Incidental macroscopic findings of pups that survived until their scheduled necropsies included small size, a bent tail and black discoloration of the 5th digit of the hindleg. Incidental macroscopic findings of pups that were found dead at the first litter check or in the days prior to the scheduled necropsy included absence of milk in the stomach and autolysis. No relationship with treatment was established for these findings and they were considered to be of no toxicological significance.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

600 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects; higest dose tested

Reproductive effects observed:

not specified

Conclusions:

In a GLP study according to OECD test guideline 422, octahydro-4,7-methano-1H-indenedimethanol (TCD Alcohol DM) did not cause either parental toxicity nor reproductive or developmental toxicity up to the highest dose tested (600 mg/kg bw/day). Parental and reproductive/developmental NOAEL was 600 mg/kg bw/day.

Executive summary:

Octahydro-4,7-methano-1H-indenedimethanol (TCD Alcohol DM) was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 150, 350 and 600 mg/kg/day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 28 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 42-53 days).

 

Formulation analysis showed that the formulations were prepared accurately and homogenously, and were stable for at least 6 hours at room temperature.

 

Parental results:

No toxicologically significant changes were noted in any of the parental parameters investigated in this study (i.e. clinical appearance, functional observations, body weights, food consumption, haematology and clinical biochemistry parameters, macroscopic examination, organ weights, and microscopic examination).

 

Reproductive/Developmental results:

No macroscopic or microscopic abnormalities were seen in the reproductive organs of the test animals indicative of reproductive toxicity of test substance. The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis. The mating, fertility and conception indices, precoital time, number of corpora lutea and implantation sites were unaffected by treatment.

 

No toxicologically relevant effects on developmental parameters were observed. The gestation index and duration of gestation were unaffected by treatment.

 

Overall, no reproductive/developmental toxicity was observed at any dose level.

 

In conclusion, treatment with TCD Alcohol DM by oral gavage in male and female Wistar Han rats at dose levels of 150, 350 and 600 mg/kg body weight/day revealed no parental toxicity up to 600 mg/kg body weight/day. No reproduction and developmental toxicity was observed for treatment up to 600 mg/kg body weight/day.

 

Based on these results, a parental, reproductive and developmental No Observed Adverse Effect Level (NOAEL) of 600 mg/kg/day was derived (van Otterdijk/NOTOX, 2010).

 

This study is acceptable and fulfils the requirements of OECD test guideline 422 for a combined repeated dose toxicity study (28 d) with the reproduction/developmental toxicity screening test.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

600 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

sufficient for assessment

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

For assessment of the reproductive and developmental toxicity of octahydro-4,7-methano-1H-indenedimethanol (TCD Alcohol DM), a screening study (OECD TG 422, combined repeated dose toxicity study (28 d) with a reproduction/developmental toxicity screening test) is available (van Otterdijk/NOTOX, 2010; RL 1, key study).

 

Octahydro-4,7-methano-1H-indenedimethanol (TCD Alcohol DM) was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 150, 350 and 600 mg/kg/day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 28 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 42-53 days).

Formulation analysis showed that the formulations were prepared accurately and homogenously, and were stable for at least 6 hours at room temperature.

Parental results:

No toxicologically significant changes were noted in any of the parental parameters investigated in this study (i.e. clinical appearance, functional observations, body weights, food consumption, haematology and clinical biochemistry parameters, macroscopic examination, organ weights, and microscopic examination).

Reproductive/Developmental results:

No macroscopic or microscopic abnormalities were seen in the reproductive organs of the test animals indicative of reproductive toxicity of test substance. The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis. The mating, fertility and conception indices, precoital time, number of corpora lutea and implantation sites were unaffected by treatment.

No toxicologically relevant effects on developmental parameters were observed. The gestation index and duration of gestation were unaffected by treatment.

Overall, no reproductive/developmental toxicity was observed at any dose level.

Based on these results, a parental, reproductive and developmental No Observed Adverse Effect Level (NOAEL) of 600 mg/kg/day was derived (van Otterdijk/NOTOX, 2010).

Short description of key information:
In a combined repeated dose toxicity study (28 d) with a reproduction/developmental toxicity screening test (OECD test guideline 422) performed under GLP with octahydro-4,7-methano-1H-indenedimethanol (TCD Alcohol DM), the reproductive NOAEL was determined to be 600 mg/kg bw/day (highest dose tested) (van Otterdijk/NOTOX, 2010).

Justification for selection of Effect on fertility via oral route:
Study specific for this endpoint. Extended one generation study or 2-generation study waived because no adverse effect noted in combined repeated dose toxicity study with the Reproduction/Developmental Toxicity Screening Test, a 90-day study nor in a developmental toxicity study.

Justification for selection of Effect on fertility via inhalation route:
Reliable data for the oral exposure route are available, thus no further study using inhalation exposure is required.

Justification for selection of Effect on fertility via dermal route:
Reliable data for the oral exposure route are available, thus no further study using inhalation exposure is required.

Effects on developmental toxicity

Description of key information

In a combined repeated dose toxicity study (28 d) with a reproduction/developmental toxicity screening test (OECD test guideline 422) performed under GLP with octahydro-4,7-methano-1H-indenedimethanol (TCD Alcohol DM), the developmental NOAEL was determined to be 600 mg/kg bw/day (highest dose tested) (van Otterdijk/NOTOX, 2010). Further, no developmental toxicity was noted in an OECD 414 study using rats up to 1000 mg/kg bw/day, the highest tested dose (Huntingdon, 2013).

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 May 2013 to 17 July 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was conducted to regulatory guidelines and in compliance with GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF, 12 Nohsan No. 8147, Agricultural Protection Bureau, November 24 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate of compliance from UM GLP Monitoring Authority included in report

Limit test:

no

Species:

rat

Strain:

other: RccHan: Wist

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: UK
- Age at study initiation: 77-86 days
- Weight at study initiation: 177 - 220
- Housing: Acclimatisation: up to four animals
During pairing: one (stock) male and one female
Gestation: one female
- Diet (e.g. ad libitum): SDS VRF1 Certified pelleted diet. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
- Water (e.g. ad libitum): Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate
intervals.
- Acclimation period: 5 days before commencement of pairing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23 deg. C
- Humidity (%): 40-70%
- Air changes (per hr): - Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hrs light

IN-LIFE DATES: From: 16 May To: 11/14 June 2013

Route of administration:

oral: gavage

Vehicle:

other: ethyl acetate/propylene glycol 16% w/w

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was suspended in ethylacetate to form a homogenous suspension. Subsequently,
propylene glycol was added under constant stirring. A series of formulations at the required concentrations was prepared by dilution of individual
weighings of the test

substance as follows:

Group Treatment Dose Nominal Volume dose Concentration of
(mg/kg/day) concentration (mL/kg) ethylacetate vehicle (w/w)
(mg/mL)
1 Control 0 0 5 16 %
2 TCD Alcohol DM 250 50 5 19 %
3 TCD Alcohol DM 500 100 5 18 %
4 TCD Alcohol DM 1000 200 5 16 %
�

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and homogeneity - Homogeneity and stability of the test material in the vehicle was demonstrated over a period of 24 hours at refrigerated storage (2-8°C); and for 15 days at ambient storage at concentrations of 1 mg/mL and 200 mg/mL as part of this programme of work in HLS Study
No. PJJ0003.
Achieved concentration - Samples of each formulation prepared for administration on Day 1 of treatment (Day 6 of gestation) and Day 14 of treatment
(Day 19 of gestation) were analysed for achieved concentration of the test substance.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1:1 with identified stock males
- Length of cohabitation: as required
- Proof of pregnancy: Ejected copulation plugs in cage tray and vaginal smears were checked for the presence of sperm, referred to as day 0 of
pregnancy
- Any other deviations from standard protocol: No

Duration of treatment / exposure:

Females were treated from Day 6 to Day 19 (inclusive) after mating, once daily at approximately the same time each day.

Frequency of treatment:

once daily

Duration of test:

20 days

Remarks:

Doses / Concentrations:
0, 50, 100, 200 mg/ml at constant dose in mg/kg/day at 5 ml/kg bw
Basis:
analytical conc.

Remarks:

Doses / Concentrations:
0, 250, 500, 1000 mg/kg bw/d
Basis:
analytical conc.

No. of animals per sex per dose:

20 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

The toxicity of TCD Alcohol DM was assessed during a Preliminary Toxicity and Embryo-Fetal Development Study by Oral Gavage Administration to Han Wistar Rats (Huntingdon Life Sciences Study No. PJJ0004, conducted at 600 and 1000 mg/kg/day). The doses were selected in conjunction with the Sponsor on the basis of data obtained in this preliminary study (HLS Study No. PJJ0004). Oral administration of dose levels of up to 1000 mg/kg/day (1000 mg/kg/day was selected as the limit dose for the OECD414 test guideline) to Han Wistar rats was tolerated without any significant signs of toxicity.

It was therefore considered that a high dose of 1000 mg/kg/day was appropriate for this embryo-fetal toxicity study. The low and intermediate doses selected were 250 and 500 mg/kg/day, respectively (applying a ratio of x2 between the dose levels).

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed physical examination was performed on each animal on Days 0, 5, 12, 18, and 20 after mating to monitor general health

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each adult was recorded on Days 0, 3 and 6-20 after mating

FOOD CONSUMPTION : Yes
The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 0-2, 3-5, 6-9, 10-13, 14-17 and
18-19 after mating inclusive

POST-MORTEM EXAMINATIONS: Yes
- Organs examined: A complete necropsy was performed in all cases.

OTHER:
- Two females receiving 1000 mg/kg/day, 4F 61 and 4F 63, were killed for welfare reasons on Days 9 and 16 respectively, due to general
poor condition.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Fetuses (live and dead) Yes

Fetal examinations:

Dissected from the uterus, individually weighed and identified within the litter using a coding system based on their position in the uterus. Examined externally with abnormalities recorded, sampled as appropriate and retained in appropriate fixative. The sex of each fetus was recorded.

Fetuses eviscerated were fixed in Industrial Methylated Spirit (IMS).
Remaining fetuses were fixed whole in Bouin’s fluid and serial sections examined for visceral abnormalities.
Processing Bouin’s fixed fetuses were subject to free-hand serial sectioning.
IMS fixed fetuses were processed and stained with Alizarin Red and assessed for skeletal development and abnormalities.

Statistics:

The following data types were analysed at each timepoint separately:
Body weight, using absolute values and gains over appropriate study periods.
Gravid uterine weight and adjusted body weight
Food consumption, over appropriate study periods
Litter size and survival indices
Fetal, placental and litter weight.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Post dose signs of salivation (with or without associated chin rubbing) were observed at all dose levels and is commonly associated with a general
distaste of formulation, but also a potential sign of reflux. Noisy breathing/rales was observed following dose administration for some individuals,
these signs are considered to be related to the oral gavage route of administration of TCD Alcohol DM, but the toxicological significance of this
finding is unclear as the signs are transient and random amongst individuals, but could be related to the route of administration of TCD Alcohol DM, rather
than direct toxicity of TCD Alcohol DM.

Treatments at 1000 mg/kg/day and to a lesser extent at 500 mg/kg/day resulted in statistically significant bodyweight loss on Days 6-7 of gestation and statistically significantly lower overall bodyweight gain, and reduced food consumption on Day 6-9 and 10-13 of gestation. The effects at 500 mg/kg/day were not considered adverse at the degree observed. The effects on bodyweight and food consumption are considered to be indicative of non-specific toxicity, with bodyweight performance showing evidence of recovery towards the end of the treatment period, and food consumption being similar to Controls towards the end of the treatment period.

Key result

Dose descriptor:

NOEL

Effect level:

500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Embryo-fetal survival, growth and development were not affected by treatment with TCD Alcohol DM at dose levels up to 1000 mg/kg/day.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other:

Remarks on result:

other: There was no effect of treatment on litter data,

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

Oral administration of octahydro-4,7-methano-1H-indenedimethanol (TCD Alcohol DM), an industrial chemical intermediate, to RccHan™;WIST (Han Wistar) rats during the organogenesis phase of gestation resulted in two deaths at 1000 mg/kg/day and overall low maternal bodyweight gain and a period of low food consumption at 1000 mg/kg/day. Similar effects on food and body weight were apparent at 500 mg/kg/day but were not considered adverse at the degree observed.
It was concluded from this study that the dosage of 500 mg/kg/day was the maternal no-observed- adverse-effect-level (NOAEL), based on slight but statistically significantly reduced food intake and body weight at the top dose, and 1000 mg/kg/day was the no-observed adverse-effect- level (NOAEL) for embryo-fetal survival, growth and development.

Executive summary:

The objective of this study was to assess the influence of TCD Alcohol DM (an industrial chemical intermediate) on embryo-fetal survival and development, when administered during the organogenesis and fetal growth phases of pregnancy in RccHanTM;WIST (Han Wistar) rats.

Three groups of 20 females received TCD Alcohol DM at doses of 250, 500 or1000 mg/kg/day by oral (gavage) administration, from Day 6 to 19 after mating. A similarly constituted Control group received the vehicle, ethylacetate in propylene glycol at the same volume dose. Animals were killed on Day 20 alter mating for reproductive assessment and fetal examination.

Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 20 alter mating and all fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination.

Results

Two females receiving 1000 mg/kg/day were killed for welfare reasons on Days 9 and 16 of gestation, due to general poor condition. Terminal clinical signs consisted of rales, piloerection and hunched posture for both animals, additionally underactive behaviour, partially closed eyelids and abnormal gait in one animal only. The common macroscopic change was the caecum contained gas, in one animal there was also gas in the stomach, jejunum and ileum, a dark liver and the rectum had no faecal pellet formation, the other animal had minimal adipose tissue. Both females were pregnant, and all implantations appeared grossly normal.

Following administration with TCD Alcohol DM, salivation and/or chin rubbing, was observed on most days during thestudy for females receiving 250, 500 or 1000 mg/kg/day. Salivation is commonly associated with general distaste to formulation. Noisy breathing and/or rales were observed in animals at all dose levels and an isolated Control animal.

Group mean statistically significant bodyweight loss was evident between Days 6-7 of gestation for females receiving 500 or 1000 mg/kg/day and for females receiving 1000 mg/kg/day statistically significant bodyweight stasis or low gain was recorded on Days 7-8 and 17-18 of gestation respectively. Overall group mean bodyweight gain (6-20) was statistically significantly low for females receiving 500 or 1000 mg/kg/day. After the weight of the gravid uterus was excluded this difference was still apparent for adjusted bodyweight and adjusted bodyweight change (6-20) at the same dose levels, however gravid uterine weights were unaffected by treatment with TCD Alcohol.

Food consumption was slightly but statistically significantly low on Days 6-9 and 10-13 of gestation for females receiving 500 or 1000 mg/kg/day.

All females were pregnant with live young on Day 20 of gestation. There was no effect of treatment on litter data as assessed by mean numbers of corpora lutea, implantations, embryo-fetal resorptions, live young, percentage sex ratio, post-implantation loss, or placental, litter and fetal weights.

Macroscopic examination of females killed on Day 20 of gestation did not reveal any findings in females receiving TCD Alcohol DM. Embryo-fetal development was also considered to be unaffected by the test material at doses up to 1000 mg/kg/day.

Conclusion

Oral administration of TCD Alcohol DM, an industrial chemical intermediate, to RccHanTM;WIST (Han Wistar) rats during the organogenesis phase of gestation resulted in two deaths at 1000 mg/kg/day and overall low maternal bodyweight gain and a period of low food consumption at 1000 mg/kg/day. Similar effects on food and body weight were apparent at 500 mg/kg/day but were not considered adverse at the degree observed.

It was concluded from this study that the dosage of 500 mg/kg/day was the maternal no-observed- adverse-effect-level (NOAEL) and 1000 mg/kg/day was the no-observed adverse-effect- level (NOAEL) for embryo-fetal survival, growth and development.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

sufficient for assessment

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

For assessment of the reproductive and developmental toxicity of octahydro-4,7-methano-1H-indenedimethanol (TCD Alcohol DM), a screening study (OECD TG 422, combined repeated dose toxicity study (28 d) with a reproduction/developmental toxicity screening test) gave a NOAEL of 600 mg/kg bw/day (van Otterdijk/NOTOX, 2010; RL 1, key study; see above: Effects on fertility - Discussion).

More recently, maternal toxicity but no developmental toxicity was seen in an OECD 414 rat study. Oral administration of ooctahydro-4,7-methano-1H-indenedimethanol (TCD Alcohol DM),

an industrial chemical intermediate, to RccHan™;WIST (Han Wistar) rats during the organogenesis phase of gestation was assessed at dose levels of 0, 250, 500 and 1000 mg/kg/day. Two females receiving 1000 mg/kg/day (4F 61 and 4F 63), were killed for welfare reasons on Days 9 and 16 respectively, due to general poor condition; rats are obligate nasal breathers and the terminal clinical signs and changes observed at necropsy were consistent with ingestion of air. The aetiology of these deaths is unknown as no corresponding pathology was performed, but reflux of dose has the potential to block nasal turbinates which can result in the ingestion of air which may have contributed to the effects observed; these observations are consistent with finding observed on animals with blocked nasal turbinates on the preliminary study (PJJ0004).

Post dose signs of salivation (with or without associated chin rubbing) were observed at all dose levels and is commonly associated with a general distaste of formulation, but also a potential sign of reflux. Noisy breathing/rales was observed following dose administration for some individuals, these signs are considered to be related to the oral gavage route of administration of TCD Alcohol DM, but the toxicological significance of this finding is unclear as the signs are transient and random amongst individuals, but could be related to the route of administration of TCD Alcohol DM, rather than direct toxicity of TCD Alcohol DM.

Treatments at 1000 mg/kg/day and to a lesser extent at 500 mg/kg/day resulted in statistically significant bodyweight loss on Days 6-7 of gestation and statistically significantly lower overall bodyweight gain, and reduced food consumption on Day 6-9 and 10-13 of gestation. The effects at 500 mg/kg/day were not considered adverse at the degree observed. The effects on bodyweight and food consumption are considered to be indicative of non-specific toxicity, with bodyweight performance showing evidence of recovery towards the end of the treatment period, and food consumption being similar to Controls towards the end of the treatment period.

It was concluded from this study that the dosage of 500 mg/kg/day was the maternal no-observed- adverse-effect-level (NOAEL), based on slight but statistically significantly reduced food intake and body weight at the top dose, and 1000 mg/kg/day was the no-observed adverse-effect- level (NOAEL) for embryo-fetal survival, growth and development Huntingdon, 2013).

Justification for selection of Effect on developmental toxicity: via oral route:
GLP guideline study of high reliability (Klimisch score 1)

Justification for selection of Effect on developmental toxicity: via inhalation route:
Reliable data for the oral exposure route are available, thus no further study using inhalation exposure is required.

Justification for selection of Effect on developmental toxicity: via dermal route:
Reliable data for the oral exposure route are available, thus no further study using inhalation exposure is required.

Justification for classification or non-classification

A reproductive and developmental NOAEL of 600 mg/kg bw/day (highest dose tested), determined in a combined repeated dose toxicity study with a reproduction/developmental toxicity screening test (OECD test guideline 422), indicates that no classification is required under Regulation (EC) No 1272/2008. There is no evidence of TCD Alcohol DM inducing adverse effects on parental fertility or development of offspring.