Registration Dossier

Administrative data

Description of key information

Repeated dose toxicity, oral: NOAEL = 10 000 ppm - corresponding to an actual test article intake of 697 and 923 mg/kg/day for males and females, respectively (OECD 422 - diet, GLP, Rel.1) - Read-across.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
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.
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)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental 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 source. 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 plastic 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.
- 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.
- 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
Details on route of administration:
The most appropriate route of administration was evaluated according to REACH Regulation. With a solubility < 1 mg/L (125-285 µg/L), the dermal uptake is likely to be low, therefore all the conditions set up in REACH Annex VIII, section 8.6.1 for testing by the dermal route are not met. In addition, the substance has a low volatility (0.128 Pa), and even if human exposure is possible via vapor, it is not considered to be the principal route of exposure. The oral route of administration via dietary inclusion was selected as it will maximize the systemic availability (internal dose).
Vehicle:
unchanged (no vehicle)
Details on oral 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.
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
Observations and examinations performed and frequency:
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 water was 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 water was 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 for animals which were sacrificed in extremis) were fasted overnight before blood sampling, but water was 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.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 7.5.1/3)
HISTOPATHOLOGY: Yes (see table 7.5.1/3)
Other examinations:
Reproduction (combined study - cf. Iuclid section 7.8.2).
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.
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.
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 eye), which was considered to be post-traumatic. These deaths were unrelated to the test item.
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
Description (incidence and severity):
Thyroid hormone analyses: Serum levels of T4 in F0-males were considered unaffected by treatment with the test item up to 10000 ppm.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant, test item-related organ weight changes were present at 10000 ppm in liver of both sexes, kidneys of males and thymus of females (cf. table 7.5.1/4 & 7.5.1/5).
In males, statistically significant, test item-related organ weight changes were present at the end of the treatment period:
• A higher liver weight, relative to body weight only, was recorded at 10000 ppm. There was no microscopic correlate for this weight increase.
• A higher kidney weight, relative to body weight only, was recorded at 10000 ppm. There was no microscopic correlate for this weight increase.
There was complete recovery for the liver and kidney weight changes after the 28-day treatment-free recovery period.
In females, statistically significant, test item-related organ weight changes were present at the end of the treatment period:
• A higher liver weight, relative to body weight only, was recorded at 10000 ppm. The microscopic correlate for this weight increase was centrilobular hepatocellular hypertrophy.
• A lower thymus weight, absolute and relative to body weight, was recorded at 10000 ppm. The microscopic correlate for this weight decrease was decreased lymphoid cellularity.
After the 28-day treatment-free recovery period there was no statistically significant organ weight change for the liver, suggesting complete recovery and there was still an apparent (but not statistically significant) lower weight for the thymus, suggesting partial recovery.
Some organ weight differences in test item-treated groups were statistically significant when compared to the concurrent control groups (testis, epididymis and female heart of Main groups, brain of females Recovery group) but were considered to be the result of a test item- related effect on final body weight (minimal in males). The statistically significant higher thyroid gland weight in males of the Recovery group at 10000 ppm was regarded to be due to a low absolute thyroid gland weight of the Recovery control males (lower absolute weight compared to the Main control group males).
There were no other test item-related organ weight changes.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test item-related red-brown discoloration was recorded for the liver of 4/10 Main females at 10000 ppm (microscopic correlate centrilobular hepatocellular hypertrophy). This finding was not present after the 28-day treatment-free period.
The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Neuropathological findings:
not examined
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 (mild-moderate). 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 these 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
Description (incidence and severity):
For reproductive and developmental results: cf. Iuclid Section 7.8.1
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:
male
Basis for effect level:
other: No adverse effect relebant for humans observed
Key result
Critical effects observed:
no

Table 7.5.1/4: 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 7.5.1/5: 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 7.5.1/6: Summary test item-related microscopic findings Males - Scheduled euthanasia animals

 

Main Males

Recovery Males

Dose level (ppm):

0

1000

3000

10000

0

10000

 

 

 

 

 

 

 

KIDNEYSa

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 7.5.1/7: Summary test item-related microscopic findings Females - Scheduled euthanasia animals

 

Main Females

Recovery Females

Dose level (ppm):

0

1000

3000

10000

0

10000

 

 

 

 

 

 

 

LIVERa

5

5

5

7

4

5

   Hypertrophy, hepatocellular,

                             centrilobular

 

 

 

 

 

 

      Minimal

-

-

-

6

-

-

 

 

 

 

 

 

 

THYMUSa

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.
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

-

0b

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.

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).

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

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.

Endpoint conclusion
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).

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

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.

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 to10% at the end of the treatment period. Additionally, nonpregnant 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).

 

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

The same conclusion applies to the target substance.

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:

The classification criteria according to the CLP and to the GHS as specific target organ toxicant (STOT) – repeated exposure, oral are not met since no significant toxic effects were observed in the combined 28 -day repeated dose toxicity study with the reproduction / development screening test performed on the source substance at the guidance value for a Category 1 classification of C < 30 mg/kg bw/day, and the guidance value for a Category 2 classification of 30 < C ≤ 300 mg/kg bw/day.

There were no data regarding the dermal and inhalation route.