Reaction Mass of Cyclohexanepropanol, 2,2,6-trimethyl-a-propyl-, (alpha.R,1R,6S)- and Cyclohexanepropanol, 2,2,6-trimethyl-a-propyl-, (alpha.S,1R,6S)-

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

From the source substance (OECD 422 / GLP / Rel.1):

- Parental NOAEL: at least 10000 ppm for females (corresponding to an actual source substance intake of 923 mg/kg/day) and 10000 ppm for males (corresponding to an actual source substance intake of 697 mg/kg/day). The hyaline droplet accumulation observed at 10000 ppm in males was considered to represent alpha-2 -microglobulin, a normal protein in male rats, which is not present in female rats nor in other mammals, including man and which is considered not adverse to humans.

- Reproduction NOAEL (Rat):at least 10000 ppm (corresponding to an actual source substance intake of 697 and 923 mg/kg/day for males and females, respectively)

- Developmental NOAEL (Rat): at least 10000 ppm (corresponding to an actual source substance intake of 697 and 923 mg/kg/day for males and females, respectively)

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From August 14, 2019 to February 12, 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD test guideline No. 422 and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Deve lopmental Toxicity Screening Test)

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

OECD GLP (inspected on 10-17 December 2019 / Signed on 24 February 2020)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han)

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/ developmental historical data in this species from the same strain and sorce. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10-11 weeks (M); 13-14 weeks (F)
- Weight at study initiation: 264-304 g (M); 189-222 g (F)
- Fasting period before study:
- Housing:
On arrival and following the pretest (females only) and pre-mating period, Main animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type, height 18 cm). Recovery males and females were housed as such during the entire study period.
During the mating phase, Main males and females were cohabitated on a 1:1 basis in Macrolon plas tic cages (MIII type, height 18 cm).
During the post-mating phase, Main males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Main Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
During the lactation phase, Main females were housed in Macrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes.
During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage enrichment, bedding material, food and water.
- Diet: Prepared diets were provided ad libitum throughout the study, except during designated procedures. During the acclimatization period, animals had free access to standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). During motor activity measurements, animals had no access to food for a maximum of 2 hours. The feed was analyzed by the supplier for nutritional components and environmental contaminants. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
- Water: Municipal tap water was freely available to each animal via water bottles. During motor activity measurements, animals had no access to water for a maximum of 2 hours. IIt is considered that there were no known contaminants in the water that would interfere with the objectives of the study.
- Acclimation period:
for 6 days prior to start of the pretest period (females) or 6 days before the commencement of administration (males).

DETAILS OF FOOD AND WATER QUALITY:
- The feed was analyzed by the supplier for nutritional components and environmental contaminants. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
- It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22
- Humidity (%): 44-72
- Air changes (per hr): > 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2019-10-08 To: 2019-12-27

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): maximum of 10 days
- Mixing appropriate amounts with (Type of food): standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Storage temperature of food: Diets were kept at room temperature for a maximum of three weeks in closed bags until use, if not used on the day of preparation. Any remaining food left after filling the food hoppers was stored at room temperature for a maximum of 10 days.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: maximum 14 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 post-coitum
- After successful mating each pregnant female was caged (how): individually housed in Macrolon plastic cages (MIII type, height 18 cm).
- Any other deviations from standard protocol:
Detection of mating was not confirmed in first instance for Female Nos. 58 and 75. Evidence of mating was by weighing the female and indirectly by delivery of a litter. Apparently, mating was overlooked in the assessment of the vaginal lavage, which explains the continued di-estrous during the mating in this female. The mating date of these animals was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post-coitum.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were performed using a validated analytical procedure.
- Concentration Analysis (all groups, each diet preparation)
Duplicate sets of samples (approximately 5 g) for each sampling time point were used for concentration analysis, the remaining samples were retained at the Test Facility as backup samples. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 20% for diet of target concentration. After acceptance of the analytical results, backup samples were discarded.
- Homogeneity Analysis (Groups 2 and 4, each diet preparation - top / middle / bottom)
Duplicate sets of samples (approximately 5 g) for each sampling time point were used for homogeneity analysis, the remaining samples were retained at the Test Facility as backup samples. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was < or = 10%. After acceptance of the analytical results, backup samples were discarded.
- Stability Analysis:
Stability analyses performed previously in conjunction with the method development and validation study demonstrated that the test item is stable in the diet when prepared and stored under the same conditions at concentrations bracketing those used in the present study.

Duration of treatment / exposure:

Minimum 28 days:
- Main males and recovery were exposed for 29 days, up to and including the day before scheduled necropsy of Main males. This included a minimum of 14 days prior to mating and during the mating period for Main males.
- Main females were exposed for 50-65 days, i.e. 14 days prior to mating (to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day of scheduled necropsy. The Main female which failed to deliver was treated for 42 days. Recovery females were treated during the same period as Main females, until at least the first scheduled necropsy of Main females (53 days).

Frequency of treatment:

Ad libitum

Dose / conc.:

1 000 ppm

Remarks:

Mean 72 and 96 kg bw/day for M and F, respectively

Dose / conc.:

3 000 ppm

Remarks:

Mean 215 and 291 kg bw/day for M and F, respectively

Dose / conc.:

10 000 ppm

Remarks:

Mean 697 and 923 kg bw/day for M and F, respectively

No. of animals per sex per dose:

Main groups: 10
Recovery groups: 5

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of a 14-day Dose Range Finder with dietary administration and in an attempt to produce graded responses to the test item.
- Rationale for animal assignment: random
- Fasting period before blood sampling for clinical biochemistry: overnight before blood sampling
- Rationale for selecting satellite groups: used to study the potential reversibility of possible toxic effects
- Post-exposure recovery period in satellite groups: 28 days

Positive control:

None

Parental animals: Observations and examinations:

MORTALITY / MORIBONDITY: Yes
- Time schedule: thoughout the study

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily, beginning during the first administration of the test item and lasting throughout the administration and recovery periods up to the day prior to necropsy.

DETAILED CLINICAL OBSERVATIONS: Yes (arena)
- Time schedule: beginning before the first administration of the test item and then once weekly throughout administration and recovery

BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of administration, and weekly thereafter. Mated Main females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. A terminal weight was recorded on the day of scheduled necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Food consumption was quantitatively measured weekly (for exceptions, see Appendix 8), except for Main males and Main females which were housed together for mating and for Main females without evidence of mating. Food consumption of mated Main females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. Scattering of food was not observed, therefore the bedding of the cages was not sieved.

WATER CONSUMPTION: Yes (qualitative)
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment / recovery period on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m.
- Anaesthetic used for blood collection: Not specified
- Animals fasted: F0-males (both Main and Recovery males) and Recovery females (except for animals which were sacrificed in extremis) were fasted overnight before blood sampling, but waterwas available. F0-Main females were not fasted overnight.
- How many animals: 5/sex/group (Main), all recovery animals
- Parameters checked in table7.5.1/2] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment / recovery period on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m.
- Animals fasted: F0-males (both Main and Recovery males) and Recovery females (except for animals which were sacrificed in extremis) were fasted overnight before blood sampling, but waterwas available. F0-Main females were not fasted overnight.
- How many animals: 5/sex/group (Main), all recovery animals
- Parameters checked in table 7.5.1/2 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule & dose groups that were examined: 5 Main males and all Recovery males during Week 4 of treatment and the selected 5 Main females during the last week of lactation (i.e. PND 6-13), and all Recovery females were tested on the first day a Main female was tested.
- Battery of functions tested:
• Hearing ability (HEARING) (Score 0 = normal/present, score 1 = abnormal/absent).
•Pupillary reflex (PUPIL L/R) (Score 0 = normal/present, score 1 = abnormal/absent).
•Static righting reflex (STATIC R) (Score 0 = normal/present, score 1 = abnormal/absent).
•Fore- and hind-limb grip strength, recorded as the mean of three measurements per animal
•Locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA). Total movements and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head.

IMMUNOLOGY: Yes
- Time schedule for collection of blood:
at the end of the treatment / recovery period on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m.
- Animals fasted: F0-males (both Main and Recovery males) and Recovery females (except foranimals which were sacrificed in extremis) were fasted overnight before blood sampling, but waterwas available. F0-Main females were not fasted overnight.
- How many animals:5/sex/group (Main), all recovery animals
- Parameters checked in table 7.5.1/2 were examined.

Oestrous cyclicity (parental animals):

Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.
Daily vaginal lavage was performed for all females (Main and Recovery) during 14 days prior to treatment (pretest period) and the first 14 days of treatment. For Main females, daily vaginal lavage was continued during mating until evidence of copulation was observed.
On the day of necropsy, a vaginal lavage was also taken from Main and Recovery females, except for animals sacrificed in extremis, to determine the stage of estrous

Sperm parameters (parental animals):

Parameters examined in [P] male parental generations: [testis weight, epididymis weight, qualitative evaluation of spermatogenesis in the testis

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: [yes/no] : yes
- 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups.

GROSS EXAMINATION OF DEAD PUPS:
Except for a few missing pups, no pups died during the course of the study.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

Postmortem examinations (parental animals):

SACRIFICE - All surviving animals
- Main Male animals: Following completion of the mating period (a minimum of 28 days of administration).
- Recovery Male animals: After the recovery period of at least 28 days, which is at least 28 days after the scheduled necropsy of Main males.
- Maternal animals which delivered: PND 14-16
- Maternal animals which failed to deliver: With evidence of mating: Post-coitum Day 27
- Recovery females: After the recovery period of at least 28 days, which is at least 28 days after the first scheduled necropsy of Main females

GROSS PATHOLOGY: Yes (see table 7.5.1/3)

HISTOPATHOLOGY: Yes (see table 7.5.1/3)

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at PND 14-16

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
- Parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
For the motor activity data set (at least 3 groups) parametric (ANOVA) tests on group means were applied with Bonferroni correction for multiple testing. Mixed modelling techniques, comparing six different covariance structures, were used in order to select the best fitting statistical model.
- Non-Parametric: Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test).
- Incidence: An overall Fisher’s exact test was used to compare all groups at the 5% significance level
. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Reproductive indices:

Mating index (%): Number of females mated / Number of females paired x 100
Precoital time: Number of days between initiation of cohabitation and confirmation of mating
Fertility index (%):Number of pregnant females / Number of females mated x 100
Gestation index (%):Number of females with living pups on Day 1 / Number of pregnant females x 100
Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

Post-implantation survival index (%): Total number of offspring born / Total number of uterine implantation sites x 100
Live birth index (%): Number of live offspring on Day 1 after littering / Total number of offspring born x 100
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

Clinical signs:

no effects observed

Description (incidence and severity):

No toxicologically relevant clinical signs were observed.
Piloerection was noted between Days 10 and 14 for several males and females of the 1000, 3000 and 10000 ppm groups and for females at 10000 ppm also between Days 31 and 50. Furthermore, hunched posture was noted for one female at 10000 ppm between Days 12 and 14. Based on the incidental occurrence, absence of a clear-dose response and full recovery during the treatment-free period, these clinical signs were considered not toxicologically relevant.
Other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment with the test item. No findings were noted during the weekly arena observations in this study.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no test item-related premature decedents in the study.
Animal No. 49 (male, Recovery at 10000 ppm) and Animal No. 63 (Recovery control female) were sacrificed for ethical reasons shortly after the retro-orbital blood sampling procedure which was performed at the end of the treatment period. Cause of morbidity for both animals was exophthalmos, further characterized by moderate hemorrhage (retro-orbital and in anterior/posterior chamber of the

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A test item-related reduced body weight gain was noted for Main females treated at 10000 ppm during the premating period and at the end of the post-coitum period (-67% and 20%, respectively).
This resulted in statistically significantly lower mean body weights at the end of the treatment period (up to 10%) when compared with concurrent controls. Mean body weights were slightly outside the range of historical control data at the end of the post-coitum and lactation phase. For Recovery females at 10000 ppm, a statistically significantly lower body weight gain was observed during the premating and mating periods when compared with Recovery controls. This resulted in a lower mean body weight (-7%) at the end of the treatment period. During the recovery period, body weight gain remained reduced when compared with Recovery controls, resulting in a 7% lower mean body weight at the end of the recovery period.
A reduced body weight gain was noted for males treated at 10000 ppm during the premating and mating periods (up to -37% when compared with controls). A normal body weight gain was noted from the second week of the recovery period onwards.
No test item-related changes in body weights and body weight gain were observed for animals of the 1000 and 3000 ppm groups.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A test item-related lower food consumption (mean of means) was noted for females at 10000 ppm when compared with concurrent controls during the premating (0.87x), mating (0.81x, Recovery females only), post-coitum (0.90x) and lactation (0.83x) periods, reaching statistical significance on 3 of 9 occasions. During the course of the Recovery period, food intake returned to normal levels. Relative food consumption for females at 10000 ppm was similar to control levels during the Treatment and Recovery Periods.
Food consumption before or after correction for body weight was similar to control levels over the treatment period for males up to 10000 ppm and females up to 3000 ppm.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No test item-related changes were noted in hematological parameters. Any statistically significant changes in hematology parameters were considered to be unrelated to treatment with the test item as these occurred in the absence of a dose-related trend.
Coagulation parameters of treated rats were considered not to have been affected by treatment with the test item. Any statistically significant changes in coagulation parameters were considered to be unrelated to treatment with the test item as these occurred in the absence of a dose-related trend.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The following changes distinguished treated from control animals at end of treatment:
- Decreased glucose concentrations in males at 10000 ppm (0.74x).
- Decreased alanine aminotransferase (ALAT) activity in females at 1000, 3000 and 10000 ppm (0.66x, 0.70x and 0.62x, respectively).
- Decreased aspartate aminotransferase (ASAT) activity in females at 1000, 3000 and 10000 ppm (0.70x, 0.76x and 0.69x, respectively).
- Decreased alkaline phosphatase (ALP) activity in females at 1000, 3000 and 10000 ppm (0.70x, 0.89x and 0.60x, respectively).
- Increased concentration of cholesterol in females at 10000 ppm (1.55x).
- Increased concentration of bile acids in females at 10000 ppm (2.64x).
- Increased concentration of calcium in females at 3000 and 10000 ppm (1.04x and 1.06x, respectively).
These alterations in clinical biochemistry parameters were considered not toxicologically relevant as mean values remained within the historical control range , based on the minimal magnitude of the change, high control values, the absence of a clear dose response and/or full recovery after a 28-day treatment free period.
Other statistically significant changes in clinical biochemistry parameters were considered to be unrelated to treatment with the test item as these occurred in the absence of a dose-related trend.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional observation parameters were considered unaffected by administration up to 10000 ppm.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals up to 10000 ppm. Grip strength and motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were noted in the kidneys of males and the liver and thymus of females (cf. Table 7.5.1/6 & 7.5.1/7).
An increased incidence and severity of hyaline droplet accumulation in the kidneys was recorded in males at the end of the 28-day treatment period at 3000 ppm (minimal-mild) and 10000 ppm (mildmoderate).
The minimal hyaline droplet accumulation recorded for the remaining groups (including controls) was regarded within background. At the end of the treatment period at 10000 ppm this was accompanied by a single incidence of granular casts (minimal). There was complete recovery for the se kidney findings after a 28-day treatment free recovery period.
Centrilobular hepatocellular hypertrophy of the liver (minimal, correlating to higher weight and red brown discoloration) was recorded in females at the end of the treatment period at 10000 ppm. There was complete recovery for this liver finding after a 28-day treatment-free recovery period.
An increased incidence/severity of decreased lymphoid cellularity of the thymus (minimal slight, correlating to lower organ weight) was recorded in females at the end of the treatment period at 10000 ppm. The minimal degree of decreased lymphoid cellularity in a few females at 1000 and 3000 ppm is considered background for lactating females in a combined 28-day study (Menke et al., 2012).
A single incidence of decreased lymphoid cellularity was present after the 28-Day treatment-free recovery period, suggesting partial recovery.
There were no other test item-related histologic changes. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain, subjected to combined 28-day toxicity study. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Length and regularity of the estrous cycle were considered not to have been affected by treatment with the test item.
Most females had regular cycles of 4 to 5 days. An irregular cycle was noted for Female No. 75 at 1000 ppm (litter with one pup) and for Female No. 60 of the control group and No. 98 at 10000 ppm the regularity of the estrous cycle was unable to determine. Given their incidental nature, and absence of a dose-related incidence, these findings did not indicate a relation with administration of the test item.

Reproductive performance:

no effects observed

Description (incidence and severity):

- Mating Index: Mating index was not affected by treatment with the test item. All females showed evidence of mating. The mating index was 100% for all groups.
- Precoital time: Precoital time was considered not to be affected by treatment with the test item. Most females showed evidence of mating within 4 days, except for one control female (No. 58) for which mating took 15 days. Given the occurrence in a control female only, this finding was considered to be unrelated to treatment with the test item.
- Number of Implantation Sites: Number of implantation sites was considered not to be affected by treatment with the test item. The mean number of implantation sites of animals treated at 1000 ppm was slightly lower when compared with controls. This could be attributed to two females at 1000 ppm (Nos. 74 and 75) who had 4 and 3 implantation sites, respectively. In absence of a dose response relationship, this was considered to be unrelated to treatment.
- Fertility Index: Fertility index not affected by treatment with the test item. All mated females were pregnant. The fertility index was 100% for all groups.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 923 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No adverse effect observed

Key result

Dose descriptor:

NOAEL

Effect level:

>= 697 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No adverse effect relevant to humans observed

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment with the test item.
For pup No. 10 from litter No. 70 at 1000 ppm, pup No 10 from litter No. 81 at 3000 ppm and pup No. 8 from litter No. 95 at 10000 ppm abnormal posture of a hindleg was noted. The nature and incidence of these and other clinical signs remained within the range considered normal for pups of this age and were therefore considered not to be related to treatment with the test item.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

- Post-Implantation Survival Index: The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment with the test item.
Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 91, 78, 93 and 87% for the control, 1000, 3000 and 10000 ppm groups, respectively.
- Litter size: Litter size was considered not affected by treatment with the test item.
Live litter sizes were 12.1, 9.4, 11.4 and 10.3 living pups/litter for the control, 1000, 3000 and 10000 groups, respectively.
- Viability index: The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was considered not affected by treatment with the test item.
Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was considered not to be affected by treatment with the test item. Viability indices were 100, 100, 99 and 98% for the control, 1000, 3000 and 10000 groups, respectively.
One pup of the 3000 ppm group and 2 pups of the 10000 ppm group were found missing on PND 3 or 4. Pups missing were most likely cannibalized. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence remained within the range considered normal for pups of this age.
- Lactation index: The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was considered not to be affected by treatment with the test item.
The lactation indices were 100, 100, 99 and 100% for the control, 1000, 3000 and 10000 ppm groups, respectively.
One pup of the 3000 ppm group was missing on PND 6. Pups missing were most likely cannibalized. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A test item-related lower body weight (up to -17% for males and females combined) was noted for pups of the 10000 ppm group on PND 7 (statistically significant for males only) and on PND 13. Mean values were below the historical control data .
Body weights of pups from the 1000 and 3000 ppm groups were considered not to be affected by treatment with the test item.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment with the test item.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Administration up to 10000 ppm had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic findings were noted among pups that were considered to be related to treatment with the test item.
The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age and were therefore considered not to be related to treatment with the test item.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

- Gestation Index and Duration: Gestation index and duration of gestation were considered not to be affected by treatment with the test item.
Except for one female at 1000 ppm (No. 74, implantations only), all pregnant females had live offspring. The gestation indices were 100, 90, 100 and 100% for the control, 1000, 3000 and 10000 groups, respectively.
The failed pregnancy of Female No.74, without related histopathology changes in reproductive organs, was judged to be unrelated to treatment with the test item due to the incidental occurrence and lack of a dose-related trend
- Parturition/Maternal Care: No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.
- Sex ratio: No toxicologically relevant effect on sex ratio was observed.
At 10000 ppm, a male female ratio of 40/60 was observed. Seven out of 10 litters had more female than male pups. As this ratio was considered within normal ranges and in absence of effects on anogenital distance, thyroid hormone levels and nipple retention, this was considered not toxicologically relevant.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

no effects observed

Description (incidence and severity):

- Clinical Biochemistry (T4 Levels): Serum T4 levels in male and female PND 14-16 pups were considered not to be affected by treatment with the test item.

Key result

Dose descriptor:

NOAEL

Remarks:

development

Generation:

F1

Effect level:

>= 10 000 ppm

Based on:

test mat.

Remarks:

697 and 923 mg/kg/day for parental males and females, respectively

Sex:

male/female

Basis for effect level:

other: No adverse effect observed

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Table 7.8.1/4: Mean percent organ weight differences from control group - Males

	Main Males					Recovery Males
Dose level (ppm):	1000		3000		10000	10000
LIVER
Absolute	-1		4		16	10
Relative to body weight	3		6		22**	7
KIDNEYS
Absolute	6		5		11	10
Relative to body weight	12		8		15**	7
*: P<0.05, **: P<0.01

Table 7.8.1/5: Mean percent organ weight difference from control group - Females

	Main Females					Recovery Females
Dose level (ppm):	1000		3000		10000	10000
LIVER
Absolute	-8		5		13	-5
Relative to body weight	-3		9		28**	3
THYMUS
Absolute	-3		-14		-35**	-20
Relative to body weight	3		-10		-27*	-14
*: P<0.05, **: P<0.01

Table 7.8.1/6: Summary test item-related microscopic findings Males - Scheduled euthanasia animals

	Main Males				Recovery Males
Dose level (ppm):	0	1000	3000	10000	0	10000
KIDNEYS^a	5	5	5	5	5	4
Hyaline droplet accumulation
Minimal	3	-	2	-	2	2
Slight	-	-	2	1	-	-
Moderate	-	-	-	4	-	-
Granular casts
Minimal	-	-	-	1	-	-

^a = Number of tissues examined from each group.

Table 7.8.1/7:Summary test item-related microscopic findings Females - Scheduled euthanasia animals

	Main Females				Recovery Females
Dose level (ppm):	0	1000	3000	10000	0	10000
LIVER^a	5	5	5	7	4	5
Hypertrophy, hepatocellular,                              centrilobular
Minimal	-	-	-	6	-	-
THYMUS^a	5	5	5	5	4	5
Decreased cellularity, lymphoid
Minimal	-	1	2	3	-	1
Slight	-	-	-	1	-	-

^a = Number of tissues examined from each group.

Conclusions:

- Parental NOAEL: at least 10000 ppm for females (corresponding to an actual test article intake of 923 mg/kg/day) and 10000 ppm for males (corresponding to an actual test article intake of 697 mg/kg/day). The hyaline droplet accumulation observed at 10000 ppm in males was considered to represent alpha-2 -microglobulin, a normal protein in male rats, which is not present in female rats nor in other mammals, including man and which is considered not adverse to humans.
- Reproduction NOAEL (Rat):at least 10000 ppm (corresponding to an actual test article intake of 697 and 923 mg/kg/day for males and females, respectively)
- Developmental NOAEL (Rat): at least 10000 ppm (corresponding to an actual test article intake of 697 and 923 mg/kg/day for males and females, respectively)

Executive summary:

In a combined repeated dose toxicity study with the reproduction / developmental screening test performed according to OECD TG No. 422 and in compliance with GLP, the test substance was administered to Wistar Han rats via diet for a minimum of 28 days, followed by a 28-day recovery period.

The recovery animals (used to study the potential reversibility of possible toxic effects) were not mated and consequently were not used for the assessment of reproduction/ developmental toxicity.

The dose levels in this study were selected to be 0, 1000, 3000, 10000 ppm, based on the results of the Dose Range Finder.

 

The study design was as follows:

 

Group No.		Test Item Identification	Dose Level (ppm)^a	Number of Animals
				Males	Females
1	Main Recovery	-	0^b	10 5	10 5
2	Main	Reaction Mass of Cyclohexanepropanol, 2,2,6-trimethyl-a-propyl-, (alpha.R,1R,6S)- and Cyclohexanepropanol, 2,2,6-trimethyl-a-propyl-, [1a(S*),6b]- (9CI)	1000	10	10
3	Main		3000	10	10
4	Main Recovery		10000	10 5	10 5

^a  The test item had a purity of 93.4%, dose calculations were not corrected for purity.

^b  Standard powder rodent diet without test item.

 

Chemical analyses of dietary preparations were conducted for each diet preparation to assess accuracy and homogeneity and dietary analyses confirmed that diets were prepared accurately and homogenously.

 

The following parameters and end points were evaluated in this study: mortality/ moribundity, clinical signs, body weight and food consumption, functional observations, estrous cycle determination, clinical pathology, measurement of thyroid hormone T4 (F0‑males), gross necropsy findings, organ weights and histopathologic examinations.

 

Parental results:

There were two animals (recovery male at 10000 ppm and recovery control female) sacrificed for ethical reasons shortly after blood sampling from the retro-orbital sinus. The cause of morbidity for both animals was exophthalmos, further characterized by moderate hemorrhage (retro-orbital and in anterior/posterior chamber of the eye), which was considered to be post-traumatic. These deaths were unrelated to the test item.

A reduced body weight gain was noted for males treated at 10000 ppm during the premating and mating periods (up to -37% when compared with controls). A normal body weight gain was noted from the second week of the recovery period onwards. The reduced body weight gain in males did not result in significantly lower absolute terminal body weights and was therefore considered not adverse.

During this study, a reduced food consumption (up to 0.83x of controls) was noted for females at 10000 ppm during the post coitum and lactation period, this resulted in a lower body weight for pregnant/lactating females up to ‑10% at the end of the treatment period. Additionally, non‑pregnant Recovery female animals had a lower body weight up to -7%. The effects of food consumption and bodyweight gain are considered likely to be related to issues of palatability as this is often observed with fragrance substances. As the body weight changes were slightly outside the range of historical control data, this would normally be considered adverse but because the reduced weight gain is associated to palatability then it is considered not adverse. However, as this is a screening study it cannot be excluded that there is a toxicity mode of action, but as there are no toxicological correlates in the biochemistry or histopathology data, then this is speculation.

Following necropsy, an increased incidence and severity of hyaline droplet accumulation was recorded in the kidneys of males at 3000 and 10000 ppm. This was considered to representalpha2uglobulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules. This male rat specific protein is not present in female rats nor in other mammals, including man. At 10000 ppm this was accompanied by granular casts, which is a degenerative alteration and therefore the kidney findings at 10000 ppm were considered to be adverse to the male rat but not relevant to humans. The slightly increased incidence and severity of hyaline droplet accumulation at 3000 ppm was not accompanied by indicators of tubular damage and was therefore considered to be non-adverse.

Minimal centrilobular hepatocellular hypertrophy was recorded in the liver of females at 10000 ppm, correlating to slightly higher relative liver weights, in the absence of any degenerative or inflammatory changes this was considered to be non-adverse.

An increased incidence/severity of decreased lymphoid cellularity in the thymus was recorded for females at 10000 ppm, which correlated with lower thymus weights. These findings can be seen as background alterations in lactating females subjected to a combined 28-day repro screening toxicity study. The minor increases in incidence and severity at 10000 ppm, in absence of any alteration in hematology parameters, were considered to be non-adverse.

No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. mortality/moribundity, clinical appearance, functional observations (motor activity, grip strength, hearing ability, pupillary reflex and static righting reflex), clinical laboratory investigations, haematology, clotting parameters and clinical biochemistry (including male T4 thyroid hormone levels).

 

Reproductive results

No reproduction toxicity was observed up to the highest dose level tested (10000 ppm).

No treatment-related significant changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle, and histopathological examination of reproductive organs).

 

Developmental results

A lower body weight was noted for pups of the 10000 ppm group on PND 7 and on PND 13, and as the values were outside the historical control range this would normally be considered adverse. However, because this effect was considered to be secondary to the reduced food consumption of the dams by palatability and therefore due to the test system, it is in this case not considered adverse. 

No toxicologically significant changes were noted in any of the other developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care, litter size and early postnatal pup development consisting of mortality, clinical signs, anogenital distance, areola/nipple retention, T4 thyroid hormone levels and macroscopic examination).

The developmental effects observed in this study (slightly reduced bodyweight gain in the pups) occurred at a dose level associated with reduced food consumption and reduced bodyweight gain in the female parents. This included a reduced food consumption during the post-coitum and lactation period and lower body weights for pregnant and lactating females at 10000 ppm. It was considered that the effects on body weight and food intake of the female parents were be caused by palatability of the diet and contributed to the observed developmental effects, particularly as there were no toxicological correlates in the biochemistry or histopathology data. However, as this is a screening study it cannot be excluded that there is an alternative mechanism for the observed effect, but this can only be speculation. No developmental effects were observed at dose levels which showed no effects on the parents.

 

In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following NOAELs were established:

- Parental NOAEL: at least 10000 ppm for females (corresponding to an actual test article intake of 923 mg/kg/day) and 10000 ppm for males (corresponding to an actual test article intake of 697 mg/kg/day). The hyaline droplet accumulation observed at 10000 ppm in males was considered to represent alpha-2 -microglobulin, a normal protein in male rats, which is not present in female rats nor in other mammals, including man and which is considered not adverse to humans.

- Reproduction NOAEL (Rat):at least 10000 ppm (corresponding to an actual test article intake of 697 and 923 mg/kg/day for males and females, respectively)

- Developmental NOAEL (Rat): at least 10000 ppm (corresponding to an actual test article intake of 697 and 923 mg/kg/day for males and females, respectively)

 

This study is acceptable and satisfies the guideline requirements for a combined 28 -day repeated dose toxicity study with the reproduction / developmental toxicity screening test (OECD 422) in rats.

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Cf. IUCLID section 13.2

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar Physico-Chemical, and Toxicological properties because of their structural similarity.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target and source substances are both multi-constituents, as reaction mass of stereoisomers of the same substance.
The target and source substances are structurally related, in that both are a reaction mass of stereoisomers of 1-(2,2,6-Trimethylcyclohexyl)hexan-3-ol. They differ by the number of constituents. The two trans 1R,6S diastereoisomers, with both 3R and 3S hydroxyl group, of the source substance are constituents of the target substance, which contains also the trans 1S,6R pair (i.e. 2 pairs of enantiomers).

3. ANALOGUE APPROACH JUSTIFICATION
Based on structural similarity (constituents of the source and the target substance are from the same pool of substance, i.e stereoisomers of 1-(2,2,6-Trimethylcyclohexyl)hexan-3-ol) it is considered appropriate and scientifically justified to read-across the data from the source to the target substance.
The study design (OECD 422, GLP) is adequate and reliable for the purpose of the prediction based on read-across. The test material used represents the source substance as described in the hypothesis in terms of purity and impurities. The results of the studies are adequate for the purpose of classification and labelling.
Therefore, based on the considerations above, it can be concluded that the result of the combined repeated dose toxicity study with the reproduction / developmental screening test conducted with the source substance is highly likely to predict the properties of the target substance and is considered as adequate to fulfil the information requirement of Annex VIII, 8.7.1.

4. DATA MATRIX
Cf. IUCLID section 13.2

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across source

Vehicle:

unchanged (no vehicle)

Clinical signs:

no effects observed

Description (incidence and severity):

No toxicologically relevant clinical signs were observed.
Piloerection was noted between Days 10 and 14 for several males and females of the 1000, 3000 and 10000 ppm groups and for females at 10000 ppm also between Days 31 and 50. Furthermore, hunched posture was noted for one female at 10000 ppm between Days 12 and 14. Based on the incidental occurrence, absence of a clear-dose response and full recovery during the treatment-free period, these clinical signs were considered not toxicologically relevant.
Other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment with the test item. No findings were noted during the weekly arena observations in this study.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no test item-related premature decedents in the study.
Animal No. 49 (male, Recovery at 10000 ppm) and Animal No. 63 (Recovery control female) were sacrificed for ethical reasons shortly after the retro-orbital blood sampling procedure which was performed at the end of the treatment period. Cause of morbidity for both animals was exophthalmos, further characterized by moderate hemorrhage (retro-orbital and in anterior/posterior chamber of the

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A test item-related reduced body weight gain was noted for Main females treated at 10000 ppm during the premating period and at the end of the post-coitum period (-67% and 20%, respectively).
This resulted in statistically significantly lower mean body weights at the end of the treatment period (up to 10%) when compared with concurrent controls. Mean body weights were slightly outside the range of historical control data at the end of the post-coitum and lactation phase. For Recovery females at 10000 ppm, a statistically significantly lower body weight gain was observed during the premating and mating periods when compared with Recovery controls. This resulted in a lower mean body weight (-7%) at the end of the treatment period. During the recovery period, body weight gain remained reduced when compared with Recovery controls, resulting in a 7% lower mean body weight at the end of the recovery period.
A reduced body weight gain was noted for males treated at 10000 ppm during the premating and mating periods (up to -37% when compared with controls). A normal body weight gain was noted from the second week of the recovery period onwards.
No test item-related changes in body weights and body weight gain were observed for animals of the 1000 and 3000 ppm groups.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A test item-related lower food consumption (mean of means) was noted for females at 10000 ppm when compared with concurrent controls during the premating (0.87x), mating (0.81x, Recovery females only), post-coitum (0.90x) and lactation (0.83x) periods, reaching statistical significance on 3 of 9 occasions. During the course of the Recovery period, food intake returned to normal levels. Relative food consumption for females at 10000 ppm was similar to control levels during the Treatment and Recovery Periods.
Food consumption before or after correction for body weight was similar to control levels over the treatment period for males up to 10000 ppm and females up to 3000 ppm.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No test item-related changes were noted in hematological parameters. Any statistically significant changes in hematology parameters were considered to be unrelated to treatment with the test item as these occurred in the absence of a dose-related trend.
Coagulation parameters of treated rats were considered not to have been affected by treatment with the test item. Any statistically significant changes in coagulation parameters were considered to be unrelated to treatment with the test item as these occurred in the absence of a dose-related trend.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The following changes distinguished treated from control animals at end of treatment:
- Decreased glucose concentrations in males at 10000 ppm (0.74x).
- Decreased alanine aminotransferase (ALAT) activity in females at 1000, 3000 and 10000 ppm (0.66x, 0.70x and 0.62x, respectively).
- Decreased aspartate aminotransferase (ASAT) activity in females at 1000, 3000 and 10000 ppm (0.70x, 0.76x and 0.69x, respectively).
- Decreased alkaline phosphatase (ALP) activity in females at 1000, 3000 and 10000 ppm (0.70x, 0.89x and 0.60x, respectively).
- Increased concentration of cholesterol in females at 10000 ppm (1.55x).
- Increased concentration of bile acids in females at 10000 ppm (2.64x).
- Increased concentration of calcium in females at 3000 and 10000 ppm (1.04x and 1.06x, respectively).
These alterations in clinical biochemistry parameters were considered not toxicologically relevant as mean values remained within the historical control range , based on the minimal magnitude of the change, high control values, the absence of a clear dose response and/or full recovery after a 28-day treatment free period.
Other statistically significant changes in clinical biochemistry parameters were considered to be unrelated to treatment with the test item as these occurred in the absence of a dose-related trend.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional observation parameters were considered unaffected by administration up to 10000 ppm.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals up to 10000 ppm. Grip strength and motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were noted in the kidneys of males and the liver and thymus of females (cf. Table 7.5.1/6 & 7.5.1/7).
An increased incidence and severity of hyaline droplet accumulation in the kidneys was recorded in males at the end of the 28-day treatment period at 3000 ppm (minimal-mild) and 10000 ppm (mildmoderate).
The minimal hyaline droplet accumulation recorded for the remaining groups (including controls) was regarded within background. At the end of the treatment period at 10000 ppm this was accompanied by a single incidence of granular casts (minimal). There was complete recovery for the se kidney findings after a 28-day treatment free recovery period.
Centrilobular hepatocellular hypertrophy of the liver (minimal, correlating to higher weight and red brown discoloration) was recorded in females at the end of the treatment period at 10000 ppm. There was complete recovery for this liver finding after a 28-day treatment-free recovery period.
An increased incidence/severity of decreased lymphoid cellularity of the thymus (minimal slight, correlating to lower organ weight) was recorded in females at the end of the treatment period at 10000 ppm. The minimal degree of decreased lymphoid cellularity in a few females at 1000 and 3000 ppm is considered background for lactating females in a combined 28-day study (Menke et al., 2012).
A single incidence of decreased lymphoid cellularity was present after the 28-Day treatment-free recovery period, suggesting partial recovery.
There were no other test item-related histologic changes. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain, subjected to combined 28-day toxicity study. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Length and regularity of the estrous cycle were considered not to have been affected by treatment with the test item.
Most females had regular cycles of 4 to 5 days. An irregular cycle was noted for Female No. 75 at 1000 ppm (litter with one pup) and for Female No. 60 of the control group and No. 98 at 10000 ppm the regularity of the estrous cycle was unable to determine. Given their incidental nature, and absence of a dose-related incidence, these findings did not indicate a relation with administration of the test item.

Reproductive performance:

no effects observed

Description (incidence and severity):

- Mating Index: Mating index was not affected by treatment with the test item. All females showed evidence of mating. The mating index was 100% for all groups.
- Precoital time: Precoital time was considered not to be affected by treatment with the test item. Most females showed evidence of mating within 4 days, except for one control female (No. 58) for which mating took 15 days. Given the occurrence in a control female only, this finding was considered to be unrelated to treatment with the test item.
- Number of Implantation Sites: Number of implantation sites was considered not to be affected by treatment with the test item. The mean number of implantation sites of animals treated at 1000 ppm was slightly lower when compared with controls. This could be attributed to two females at 1000 ppm (Nos. 74 and 75) who had 4 and 3 implantation sites, respectively. In absence of a dose response relationship, this was considered to be unrelated to treatment.
- Fertility Index: Fertility index not affected by treatment with the test item. All mated females were pregnant. The fertility index was 100% for all groups.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 923 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No adverse effect observed

Key result

Dose descriptor:

NOAEL

Effect level:

>= 697 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No adverse effect relevant to humans observed

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment with the test item.
For pup No. 10 from litter No. 70 at 1000 ppm, pup No 10 from litter No. 81 at 3000 ppm and pup No. 8 from litter No. 95 at 10000 ppm abnormal posture of a hindleg was noted. The nature and incidence of these and other clinical signs remained within the range considered normal for pups of this age and were therefore considered not to be related to treatment with the test item.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

- Post-Implantation Survival Index: The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment with the test item.
Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 91, 78, 93 and 87% for the control, 1000, 3000 and 10000 ppm groups, respectively.
- Litter size: Litter size was considered not affected by treatment with the test item.
Live litter sizes were 12.1, 9.4, 11.4 and 10.3 living pups/litter for the control, 1000, 3000 and 10000 groups, respectively.
- Viability index: The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was considered not affected by treatment with the test item.
Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was considered not to be affected by treatment with the test item. Viability indices were 100, 100, 99 and 98% for the control, 1000, 3000 and 10000 groups, respectively.
One pup of the 3000 ppm group and 2 pups of the 10000 ppm group were found missing on PND 3 or 4. Pups missing were most likely cannibalized. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence remained within the range considered normal for pups of this age.
- Lactation index: The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was considered not to be affected by treatment with the test item.
The lactation indices were 100, 100, 99 and 100% for the control, 1000, 3000 and 10000 ppm groups, respectively.
One pup of the 3000 ppm group was missing on PND 6. Pups missing were most likely cannibalized. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A test item-related lower body weight (up to -17% for males and females combined) was noted for pups of the 10000 ppm group on PND 7 (statistically significant for males only) and on PND 13. Mean values were below the historical control data .
Body weights of pups from the 1000 and 3000 ppm groups were considered not to be affected by treatment with the test item.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment with the test item.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Administration up to 10000 ppm had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic findings were noted among pups that were considered to be related to treatment with the test item.
The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age and were therefore considered not to be related to treatment with the test item.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

- Gestation Index and Duration: Gestation index and duration of gestation were considered not to be affected by treatment with the test item.
Except for one female at 1000 ppm (No. 74, implantations only), all pregnant females had live offspring. The gestation indices were 100, 90, 100 and 100% for the control, 1000, 3000 and 10000 groups, respectively.
The failed pregnancy of Female No.74, without related histopathology changes in reproductive organs, was judged to be unrelated to treatment with the test item due to the incidental occurrence and lack of a dose-related trend
- Parturition/Maternal Care: No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.
- Sex ratio: No toxicologically relevant effect on sex ratio was observed.
At 10000 ppm, a male female ratio of 40/60 was observed. Seven out of 10 litters had more female than male pups. As this ratio was considered within normal ranges and in absence of effects on anogenital distance, thyroid hormone levels and nipple retention, this was considered not toxicologically relevant.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

no effects observed

Description (incidence and severity):

- Clinical Biochemistry (T4 Levels): Serum T4 levels in male and female PND 14-16 pups were considered not to be affected by treatment with the test item.

Key result

Dose descriptor:

NOAEL

Remarks:

development

Generation:

F1

Effect level:

>= 10 000 ppm

Based on:

test mat.

Remarks:

697 and 923 mg/kg/day for parental males and females, respectively

Sex:

male/female

Basis for effect level:

other: No adverse effect observed

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Table 7.8.1/4: Mean percent organ weight differences from control group - Males

	Main Males					Recovery Males
Dose level (ppm):	1000		3000		10000	10000
LIVER
Absolute	-1		4		16	10
Relative to body weight	3		6		22**	7
KIDNEYS
Absolute	6		5		11	10
Relative to body weight	12		8		15**	7
*: P<0.05, **: P<0.01

Table 7.8.1/5: Mean percent organ weight difference from control group - Females

	Main Females					Recovery Females
Dose level (ppm):	1000		3000		10000	10000
LIVER
Absolute	-8		5		13	-5
Relative to body weight	-3		9		28**	3
THYMUS
Absolute	-3		-14		-35**	-20
Relative to body weight	3		-10		-27*	-14
*: P<0.05, **: P<0.01

Table 7.8.1/6: Summary test item-related microscopic findings Males - Scheduled euthanasia animals

	Main Males				Recovery Males
Dose level (ppm):	0	1000	3000	10000	0	10000
KIDNEYS^a	5	5	5	5	5	4
Hyaline droplet accumulation
Minimal	3	-	2	-	2	2
Slight	-	-	2	1	-	-
Moderate	-	-	-	4	-	-
Granular casts
Minimal	-	-	-	1	-	-

^a = Number of tissues examined from each group.

Table 7.8.1/7:Summary test item-related microscopic findings Females - Scheduled euthanasia animals

	Main Females				Recovery Females
Dose level (ppm):	0	1000	3000	10000	0	10000
LIVER^a	5	5	5	7	4	5
Hypertrophy, hepatocellular,                              centrilobular
Minimal	-	-	-	6	-	-
THYMUS^a	5	5	5	5	4	5
Decreased cellularity, lymphoid
Minimal	-	1	2	3	-	1
Slight	-	-	-	1	-	-

^a = Number of tissues examined from each group.

Conclusions:

- Parental NOAEL: at least 10000 ppm for females (corresponding to an actual source substance intake of 923 mg/kg/day) and 10000 ppm for males (corresponding to an actual source substance intake of 697 mg/kg/day). The hyaline droplet accumulation observed at 10000 ppm in males was considered to represent alpha-2 -microglobulin, a normal protein in male rats, which is not present in female rats nor in other mammals, including man and which is considered not adverse to humans.
- Reproduction NOAEL (Rat):at least 10000 ppm (corresponding to an actual source substance of 697 and 923 mg/kg/day for males and females, respectively)
- Developmental NOAEL (Rat): at least 10000 ppm (corresponding to an actual source substance intake of 697 and 923 mg/kg/day for males and females, respectively).
The same conclusions apply to the target substance.

Executive summary:

In a combined repeated dose toxicity study with the reproduction / developmental screening test performed according to OECD TG No. 422 and in compliance with GLP, the source substance was administered to Wistar Han rats via diet for a minimum of 28 days, followed by a 28-day recovery period.

The recovery animals (used to study the potential reversibility of possible toxic effects) were not mated and consequently were not used for the assessment of reproduction/ developmental toxicity.

The dose levels in this study were selected to be 0, 1000, 3000, 10000 ppm, based on the results of the Dose Range Finder.

 

The study design was as follows:

 

Group No.		Test Item Identification	Dose Level (ppm)^a	Number of Animals
				Males	Females
1	Main Recovery	-	0^b	10 5	10 5
2	Main	Reaction Mass of Cyclohexanepropanol, 2,2,6-trimethyl-a-propyl-, (alpha.R,1R,6S)- and Cyclohexanepropanol, 2,2,6-trimethyl-a-propyl-, [1a(S*),6b]- (9CI)	1000	10	10
3	Main		3000	10	10
4	Main Recovery		10000	10 5	10 5

^a  The test item had a purity of 93.4%, dose calculations were not corrected for purity.

^b  Standard powder rodent diet without test item.

 

Chemical analyses of dietary preparations were conducted for each diet preparation to assess accuracy and homogeneity and dietary analyses confirmed that diets were prepared accurately and homogenously.

 

The following parameters and end points were evaluated in this study: mortality/ moribundity, clinical signs, body weight and food consumption, functional observations, estrous cycle determination, clinical pathology, measurement of thyroid hormone T4 (F0‑males), gross necropsy findings, organ weights and histopathologic examinations.

 

Parental results:

There were two animals (recovery male at 10000 ppm and recovery control female) sacrificed for ethical reasons shortly after blood sampling from the retro-orbital sinus. The cause of morbidity for both animals was exophthalmos, further characterized by moderate hemorrhage (retro-orbital and in anterior/posterior chamber of the eye), which was considered to be post-traumatic. These deaths were unrelated to the test item.

A reduced body weight gain was noted for males treated at 10000 ppm during the premating and mating periods (up to -37% when compared with controls). A normal body weight gain was noted from the second week of the recovery period onwards. The reduced body weight gain in males did not result in significantly lower absolute terminal body weights and was therefore considered not adverse.

During this study, a reduced food consumption (up to 0.83x of controls) was noted for females at 10000 ppm during the post coitum and lactation period, this resulted in a lower body weight for pregnant/lactating females up to ‑10% at the end of the treatment period. Additionally, non‑pregnant Recovery female animals had a lower body weight up to -7%. The effects of food consumption and bodyweight gain are considered likely to be related to issues of palatability as this is often observed with fragrance substances. As the body weight changes were slightly outside the range of historical control data, this would normally be considered adverse but because the reduced weight gain is associated to palatability then it is considered not adverse. However, as this is a screening study it cannot be excluded that there is a toxicity mode of action, but as there are no toxicological correlates in the biochemistry or histopathology data, then this is speculation.

Following necropsy, an increased incidence and severity of hyaline droplet accumulation was recorded in the kidneys of males at 3000 and 10000 ppm. This was considered to representalpha2uglobulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules. This male rat specific protein is not present in female rats nor in other mammals, including man. At 10000 ppm this was accompanied by granular casts, which is a degenerative alteration and therefore the kidney findings at 10000 ppm were considered to be adverse to the male rat but not relevant to humans. The slightly increased incidence and severity of hyaline droplet accumulation at 3000 ppm was not accompanied by indicators of tubular damage and was therefore considered to be non-adverse.

Minimal centrilobular hepatocellular hypertrophy was recorded in the liver of females at 10000 ppm, correlating to slightly higher relative liver weights, in the absence of any degenerative or inflammatory changes this was considered to be non-adverse.

An increased incidence/severity of decreased lymphoid cellularity in the thymus was recorded for females at 10000 ppm, which correlated with lower thymus weights. These findings can be seen as background alterations in lactating females subjected to a combined 28-day repro screening toxicity study. The minor increases in incidence and severity at 10000 ppm, in absence of any alteration in hematology parameters, were considered to be non-adverse.

No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. mortality/moribundity, clinical appearance, functional observations (motor activity, grip strength, hearing ability, pupillary reflex and static righting reflex), clinical laboratory investigations, haematology, clotting parameters and clinical biochemistry (including male T4 thyroid hormone levels).

 

Reproductive results

No reproduction toxicity was observed up to the highest dose level tested (10000 ppm).

No treatment-related significant changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle, and histopathological examination of reproductive organs).

 

Developmental results

A lower body weight was noted for pups of the 10000 ppm group on PND 7 and on PND 13, and as the values were outside the historical control range this would normally be considered adverse. However, because this effect was considered to be secondary to the reduced food consumption of the dams by palatability and therefore due to the test system, it is in this case not considered adverse. 

No toxicologically significant changes were noted in any of the other developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care, litter size and early postnatal pup development consisting of mortality, clinical signs, anogenital distance, areola/nipple retention, T4 thyroid hormone levels and macroscopic examination).

The developmental effects observed in this study (slightly reduced bodyweight gain in the pups) occurred at a dose level associated with reduced food consumption and reduced bodyweight gain in the female parents. This included a reduced food consumption during the post-coitum and lactation period and lower body weights for pregnant and lactating females at 10000 ppm. It was considered that the effects on body weight and food intake of the female parents were be caused by palatability of the diet and contributed to the observed developmental effects, particularly as there were no toxicological correlates in the biochemistry or histopathology data. However, as this is a screening study it cannot be excluded that there is an alternative mechanism for the observed effect, but this can only be speculation. No developmental effects were observed at dose levels which showed no effects on the parents.

 

In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following NOAELs were established:

- Parental NOAEL: at least 10000 ppm for females (corresponding to an actual source substance intake of 923 mg/kg/day) and 10000 ppm for males (corresponding to an actual source substance intake of 697 mg/kg/day). The hyaline droplet accumulation observed at 10000 ppm in males was considered to represent alpha-2 -microglobulin, a normal protein in male rats, which is not present in female rats nor in other mammals, including man and which is considered not adverse to humans.

- Reproduction NOAEL (Rat):at least 10000 ppm (corresponding to an actual source substance intake of 697 and 923 mg/kg/day for males and females, respectively)

- Developmental NOAEL (Rat): at least 10000 ppm (corresponding to an actual source substance intake of 697 and 923 mg/kg/day for males and females, respectively)

The same conclusions apply to the target substance. Refer to IUCLID Section 11 for read-across justification.

 

This study is acceptable and satisfies the guideline requirements for a combined 28 -day repeated dose toxicity study with the reproduction / developmental toxicity screening test (OECD 422) in rats.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

697 mg/kg bw/day

Study duration:

subacute

Experimental exposure time per week (hours/week):

168

Species:

rat

Quality of whole database:

The key study is GLP-compliant and of high quality (Klimisch score = 1).

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

No study was available on the substance itself, therefore a read-across approach was used. The source substance is considered adequate for read-across purposes as the data relates to a mixture composed of the same isomers that the target substance, but at different ratios (see Iuclid section 13 for additional justification).

A key study was available on the source substance (CRL, 2020, Rel. 1). In this combined repeated dose toxicity study with the reproduction / developmental screening test performed according to OECD TG No. 422 and in compliance with GLP, the test substance was administered to Wistar Han rats via diet for a minimum of 28 days, followed by a 28-day recovery period. The recovery animals were not mated and consequently were not used for the assessment of reproduction/ developmental toxicity. The dose levels in this study were selected to be 0, 1000, 3000, 10000 ppm.

 

Parental results:

There were two animals (recovery male at 10000 ppm and recovery control female) sacrificed for ethical reasons shortly after blood sampling from the retro-orbital sinus. The cause of morbidity for both animals was exophthalmos, further characterized by moderate hemorrhage (retro-orbital and in anterior/posterior chamber of the eye), which was considered to be post-traumatic. These deaths were unrelated to the test item.

A reduced body weight gain was noted for males treated at 10000 ppm during the premating and mating periods (up to -37% when compared with controls). A normal body weight gain was noted from the second week of the recovery period onwards. The reduced body weight gain in males did not result in significantly lower absolute terminal body weights and was therefore considered not adverse.

During this study, a reduced food consumption (up to 0.83x of controls) was noted for females at 10000 ppm during the post coitum and lactation period, this resulted in a lower body weight for pregnant/lactating females up to ‑10% at the end of the treatment period. Additionally, non‑pregnant Recovery female animals had a lower body weight up to -7%. The effects of food consumption and bodyweight gain are considered likely to be related to issues of palatability as this is often observed with fragrance substances. As the body weight changes were slightly outside the range of historical control data, this would normally be considered adverse but because the reduced weight gain is associated to palatability then it is considered not adverse. However, as this is a screening study it cannot be excluded that there is a toxicity mode of action, but as there are no toxicological correlates in the biochemistry or histopathology data, then this is speculation.

Following necropsy, an increased incidence and severity of hyaline droplet accumulation was recorded in the kidneys of males at 3000 and 10000 ppm. This was considered to representalpha2uglobulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules. This male rat specific protein is not present in female rats nor in other mammals, including man. At 10000 ppm this was accompanied by granular casts, which is a degenerative alteration and therefore the kidney findings at 10000 ppm were considered to be adverse to the male rat but not relevant to humans. The slightly increased incidence and severity of hyaline droplet accumulation at 3000 ppm was not accompanied by indicators of tubular damage and was therefore considered to be non-adverse.

Minimal centrilobular hepatocellular hypertrophy was recorded in the liver of females at 10000 ppm, correlating to slightly higher relative liver weights, in the absence of any degenerative or inflammatory changes this was considered to be non-adverse.

An increased incidence/severity of decreased lymphoid cellularity in the thymus was recorded for females at 10000 ppm, which correlated with lower thymus weights. These findings can be seen as background alterations in lactating females subjected to a combined 28-day repro screening toxicity study. The minor increases in incidence and severity at 10000 ppm, in absence of any alteration in hematology parameters, were considered to be non-adverse.

No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. mortality/moribundity, clinical appearance, functional observations (motor activity, grip strength, hearing ability, pupillary reflex and static righting reflex), clinical laboratory investigations, haematology, clotting parameters and clinical biochemistry (including male T4 thyroid hormone levels).

 

Reproductive results

No reproduction toxicity was observed up to the highest dose level tested (10000 ppm).

No treatment-related significant changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle, and histopathological examination of reproductive organs).

 

Developmental results

A lower body weight was noted for pups of the 10000 ppm group on PND 7 and on PND 13, and as the values were outside the historical control range this would normally be considered adverse. However, because this effect was considered to be secondary to the reduced food consumption of the dams by palatability and therefore due to the test system, it is in this case not considered adverse. 

No toxicologically significant changes were noted in any of the other developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care, litter size and early postnatal pup development consisting of mortality, clinical signs, anogenital distance, areola/nipple retention, T4 thyroid hormone levels and macroscopic examination).

The developmental effects observed in this study (slightly reduced bodyweight gain in the pups) occurred at a dose level associated with reduced food consumption and reduced bodyweight gain in the female parents. This included a reduced food consumption during the post-coitum and lactation period and lower body weights for pregnant and lactating females at 10000 ppm. It was considered that the effects on body weight and food intake of the female parents were be caused by palatability of the diet and contributed to the observed developmental effects, particularly as there were no toxicological correlates in the biochemistry or histopathology data. However, as this is a screening study it cannot be excluded that there is an alternative mechanism for the observed effect, but this can only be speculation. No developmental effects were observed at dose levels which showed no effects on the parents.

 

In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following NOAELs were established:

- Parental NOAEL: at least 10000 ppm for females (corresponding to an actual source substance intake of 923 mg/kg/day) and 10000 ppm for males (corresponding to an actual source substance intake of 697 mg/kg/day). The hyaline droplet accumulation observed at 10000 ppm in males was considered to represent alpha-2 -microglobulin, a normal protein in male rats, which is not present in female rats nor in other mammals, including man and which is considered not adverse to humans.

- Reproduction NOAEL (Rat):at least 10000 ppm (corresponding to an actual source substance intake of 697 and 923 mg/kg/day for males and females, respectively)

- Developmental NOAEL (Rat): at least 10000 ppm (corresponding to an actual source substance intake of 697 and 923 mg/kg/day for males and females, respectively)

Effects on developmental toxicity

Description of key information

This study is not required at REACh Annex VIII level.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Harmonised classification:

The substance has no harmonised classification according to the Regulation (EC) No 1272/2008 (CLP).

 

Self-classification:

Based on the available information, no self-classification is proposed according to the CLP and to the GHS.