4-Decenal, 9-hydroxy-5,9-dimethyl-

Endpoint summary

Description of key information

NOAEL = 800 mg/kg/day for males or females (OECD 407, GLP, K, rel. 1)

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:

08 August 2013 to 28 February 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in compliance with OECD Guideline No. 407 without any deviation.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted 3 October 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

May 30, 2008

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Wistar Han™:RccHan™:WIST strain

Details on species / strain selection:

The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Age at study initiation: ca. 7 weeks
- Weight at study initiation: Males: 179 to 228 g (mean 210 g), females: 122 to 148 g (mean 133 g)
- Housing: Animals were housed in groups of five in Makrolon type-4 cages with wire mesh tops and standard softwood bedding (J. Rettenmaier & Söhne GmbH & Co. KG, 73494 Rosenberg / Germany, imported by Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) including paper enrichment (Enviro-dri from Lillico, Biotechnology, Surrey / UK).
- Diet: Pelleted standard Harlan Teklad 2914C (batch no. 29/13) rat / mouse maintenance diet (Provimi Kliba AG, 4303 Kaiseraugst / Switzerland), ad libitum
- Water: Community tap-water from Itingen, ad libitum in water bottles
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 ºC
- Humidity: 30-70 %
- Air changes: 10-15 changes/ h
- Photoperiod: 12 h dark/ 12 h light
- Music: at least eight hours music during the light period.
Values outside of these ranges occasionally occurred on one day for humidity, probably following room cleaning, and are considered not to have any influence on the study.

IN-LIFE DATES: 08 August 2013 to 26 September 2013

Route of administration:

oral: gavage

Details on route of administration:

The test item was administered daily, for twenty-eight consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test item was prepared, weekly, at the appropriate concentrations as a solution in corn oil (Carl Roth GmbH & Co. KG, Schoemperlenstr. 3-5 76185 Karlsruhe / Germany Batch Number: 382191749). The test item was weighed into a tared glass container on a suitable precision balance and the vehicle, corn oil, was added to give the appropriate final concentration of the test item in the suspension. The mixtures were stirred using a magnetic stirrer and used at room temperature (15 - 25 °C).
Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

The stability of the test item formulations were based upon the results of stability analyses performed during Harlan Laboratories study D77427 (non-GLP): at least 4 hours at room temperature (20 ± 5 °C) or 8 days in the refrigerator (2 ± 8 °C). Formulations were therefore prepared weekly during the treatment period and stored in a refrigerator (2 ± 8 °C) in glass beakers.

VEHICLE
- Concentration in vehicle: 10, 40 and 160 mg/mL/day
- Amount of vehicle (if gavage): 5 mL/kg bw

DOSE VOLUME: 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The dose formulations were analyzed using a GC-FID method provided by the Sponsor and adapted by Harlan Laboratories Ltd. The samples (generally 1 g each) were delivered to the analytical laboratory. Concentration, homogeneity and stability of dose formulations were determined in samples taken after experimental start.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

5 animals/sex for the intermediate dose groups (Group 2 and 3) and 10 animals/sex for the controls and the high dose groups (Group 1 and 4)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on a previous dose range finding toxicity study in Wistar rats, Harlan Laboratories study D77427 (non-GLP).

- Rationale for animal assignment: Animals were randomly allocated to treatment groups using a stratified body weight randomisation procedure and the group mean body weights were then determined to ensure similarity between the treatment groups.

The test item was administered daily to Wistar rats at dose levels of 50, 200 and 800 mg/kg body weight/day and a control group was treated similarly with the vehicle, corn oil, only. The groups comprised 5 animals per sex which were sacrificed after 28 days of treatment, and additional 5 rats per sex and group were used at 0 and 800 mg/kg/day. These animals were treated for 28 days and then allowed a 14-day treatment-free recovery period after which they were sacrificed.

Positive control:

Not applicable

Observations and examinations performed and frequency:

MORTALITY/VIABILITY
Observations for mortality/viability were recorded twice daily.

OBSERVATIONS
- DAILY OBSERVATIONS: The animals were observed for clinical signs once daily before commencement of administration; twice daily on day 1; three times on days 2 and 3; twice daily thereafter and once daily during the recovery phase.
- WEEKLY BEHAVORIAL OBSERVATIONS: The animals were observed once weekly before commencement of administration and during the study (weeks 1-3). The observation was not performed during the recovery phase.
- FUNCTIONAL OBSERVATIONAL BATTERY:
During the last week of treatment (Week 4: 10 Sept 2013), relevant parameters from a modified Irwin screen test were performed on all rats in place of the usual weekly behavioral observation. The animals were observed in their home cages, outside their home cages in a standard arena and in the hand. Any abnormal findings were recorded and graded in severity.
Hind- and forelimb grip strength measurement was performed using a push-pull strain gauge
(Mecmesin, AFG 25N).
Locomotor activity was measured quantitatively. Decreased or increased activity was recorded.
Activity was measured with an AMS Föhr Medical Instruments GmbH (FMI) and DeMeTec GmbH. Activity of the animals was recorded for 10-minute intervals over a period of 60 minutes.
These data and the total activity over 60 minutes were reported.

BODY WEIGHT:
Animals were weighed weekly during pre-randomization, acclimatization, treatment and recovery periods and before necropsy.

FOOD AND WATER CONSUMPTION :
The food consumption was recorded weekly during the acclimatization, treatment and recovery periods. Water consumption was not recorded.

CLINICAL PATHOLOGY : HAEMATOLOGY & CLINICAL CHEMISTRY
- Time schedule for collection of blood: After 4 weeks and 6 weeks from sublingual vein for blood samples.
- Anaesthetic used for blood collection: isoflurane anaesthesia
- How many animals: All the animals, after fasting period of 18 hours, early in the working day to reduce biological variation caused by circadian rhythms (access to water ad libitum).
Urine was collected during the fasting period into a specimen vial, using a metabolism cage.
- Parameters checked in table [7.5.1/1] were examined.

Sacrifice and pathology:

GROSS PATHOLOGY:
All animals were weighed and necropsied after 4 weeks and 6 weeks (recovery). Descriptions of all macroscopic abnormalities were recorded. All animals surviving to the end of the observation period were anesthetized by
intraperitoneal injection of pentobarbitone and killed by exsanguination.
Samples of tissues and organs reported on the table 7.5.1/2 were collected from all animals at necropsy and fixed in neutral phosphate buffered 4 % formaldehyde solution (unless otherwise indicated).
The organ to terminal body weight ratios as well as organ to brain weight ratios were determined.
The determination of the terminal body weight was performed immediately prior to necropsy.

HISTOPATHOLOGY:
All organ and tissue samples were processed, embedded and cut at an approximate thickness of 2 to 4 micrometers, and stained with hematoxylin and eosin.
Slides of all organs and tissues listed in boldface type (see table 7.5.1/2) that were collected at scheduled sacrifice from the animals of all control and high-dose groups were examined by a pathologist.
The same applies to all occurring gross lesions and to the control animal, which died spontaneously. The stage of estrus was also evaluated during examination of the vagina and reported in the pathology report.
Since test item-related morphologic changes were detected in livers of both sexes and kidneys of males of the high-dose group, the livers of males and females and kidneys of males from the mid- and low-dose group animals were examined to establish a no-effect level.
The examination was carried out on the tissues reported on the table 7.5.1/2.

Statistics:

The following statistical methods were used to analyze the grip strength, locomotor activity, body weight, macroscopic findings, organ weights and ratios, as well as clinical laboratory data:
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

- DAILY OBSERVATIONS: No clinical signs were noted the control group and in females treated with 50 mg/kg/day (group 2). At 50 mg/kg/day, only one male showed slight to moderate salivation after application on days 15 and 27 only.
At 200 mg/kg/day (group 3), salivation after application was noted at a lower incidence and grade - in four of the five males and one female on single to few days at slight severity.
At 800 mg/kg/day (group 4), all animals showed slight to moderate salivation after dosing on most treatment days, partially with reddish discoloration. One male additionally had bedding in the mouth on day 1. Salivation was also noted in four females at the time of daily observation on day 28 of treatment. One further female of this group showed ruffled fur transiently on days 6 and 7 of treatment.

- WEEKLY BEHAVIORAL OBSERVATIONS: No additional clinical signs were noted during weekly behavioral observations.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One control animal (female no. 38) died at the end of the recovery period on the scheduled day of necropsy after blood sampling. This death was attributed to the blood sampling procedure. All other animals survived the scheduled treatment or recovery period.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

- No test item-related differences in body weights or body weight gain between control and test item-treated groups were noted.
- Slightly lower body weight gain in males treated with 800 mg/kg/day on day 8 of treatment was considered not to be toxicologically relevant as the mean body weight showed no difference and the statistical significance was low. Increased body weight gain compared to the control group on days 8 and 14 of the recovery phase in males treated with 800 mg/kg/day was considered to be incidental as there was no corresponding finding in females or clear contrary tendency during treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No test item-related effects on absolute or relative food consumption were noted. The amount of feed consumed by test item-treated animals and control animals was comparable.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The following statistically significant differences between test item-treated and control animals at the end of treatment were considered to be possibly test item-related as they were noted in the high dose group but in one sex only:
• Slightly increased partial thromboplastin time (+9%, p<0.05) in males treated with 800 mg/kg/day
• Slightly increased hemoglobin concentration distribution width (+8%, p<0.05 and +12%, p<0.01) in females treated with 200 or 800 mg/kg/day.

Both changes were still present in males and females treated with 800 mg/kg/day, respectively, at the end of the recovery period. However, due to the low magnitude of the changes and since the values were within the range of historical control data for rats of this strain and age, these changes were considered to be non-adverse.
All further statistically significant differences to the control group were noted in the low or mid dose group of one gender or after the recovery period only and were therefore considered not to be related to treatment with the test item. Decreased methemoglobin in females treated with 800 mg/kg/day at the end of treatment was considered not to be relevant.

CONCLUSION: Non-adverse effects

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The following statistically significant differences between test item-treated and control animals at the end of treatment were considered to be possibly test item-related as they were noted in the high dose group but in one sex only:
• Slightly increased glucose levels (+19%, p<0.05) in males treated with 800 mg/kg/day
• Slightly increased creatinine concentration (+12%, p<0.05) in females treated with 800 mg/kg/day
• Slightly increased triglyceride (+41%, p<0.05) and phospholipid (+16%, p<0.05) concentrations in females treated with 800 mg/kg/day
• Slightly increased alkaline phosphatase activity (+47%, p<0.05) in females treated with 800 mg/kg/day
• Slightly increased sodium concentration (+3%, p<0.01) in females treated with 800 mg/kg/day
• Slightly increased potassium concentration (+11%, p<0.05 and +14%, p<0.01) in males treated with 200 or 800 mg/kg/day
• Slightly increased phosphorus concentration (+19%, p<0.01) in females treated with 800 mg/kg/day
• Slightly increased albumin concentration (+8%, p<0.05 and +9%, p<0.01) in females treated with 200 or 800 mg/kg/day and increased albumin/globulin ratio (+16%, p<0.01) in females treated with 800 mg/kg/day
• Slightly decreased globulin concentration (-10%, p<0.01) and increased albumin/globulin ratio (+12%, p<0.05) in males treated with 800 mg/kg/day

Of these mentioned results, only glucose levels in males and sodium concentration, albumin concentration and albumin/globulin ratio in females were out of the range of historical control data for rats of this strain and age. However, results for control animals were quite high or out of this range as well and no similar changes were present at the end of the recovery period. Thus, the listed changes were considered not to be adverse.

Decreased total bilirubin and creatine kinase concentrations in males treated with 800 mg/kg/day were considered not to be biologically relevant.
At the end of the recovery period, slightly increased globulin concentration (+12%, p<0.01) and decreased albumin/globulin ratio (-9%, p<0.05) in males treated with 800 mg/kg/day may be considered a compensatory reaction to the previously noted changes.
Decreased lactate dehydrogenase activity and increased protein concentration in males treated with 800 mg/kg/day were considered not to be test item-related as no corresponding changes were noted at the end of treatment.

CONCLUSION: Non-adverse effects

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related findings in urinalysis parameters were noted.
Decreased urine volume together with increased relative density in males treated with 800 mg/kg/day at the end of the recovery period were considered to be incidental as there was no corresponding finding in females or at the end of the treatment period.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

- FUNCTIONAL OBSERVATIONAL BATTERY: No additional clinical signs were noted during functional observational battery in week 4.

Increased mean fore and hind limb grip strength was noted in males at all dose levels with the difference to controls attaining statistical significance except for hind limb grip strength in males treated with 200 mg/kg/day. The difference to controls was neither dose-related nor generally associated with toxicological relevance. Therefore, this finding was considered to be incidental.

Increased locomotor activity in females treated with 200 or 800 mg/kg/day at 0-10 minutes was a finding restricted to females and to these first minutes and was considered to be incidental.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

After 28 days of treatment, differences in organ weights between test item-treated rats and the vehicle control group were noted and reported in the Table 7.5.1/3 thereafter.

At the end of treatment, a dose-related increase in mean absolute and relative liver weights noted in males and females of all test item-treated groups was considered to be test item-related. The difference to controls attained statistical significance in animals treated with 800 or 200 mg/kg/day.
Additionally, decreased mean absolute and relative adrenal weights were noted in males treated with 800 mg/kg/day and increased absolute and relative kidney weights were noted in females treated with 800 mg/kg/day. These changes were considered to be test item-related as well because the respective change was noted consistently in absolute and relative weights.
Consequently, increased kidney to brain weight ratio in females treated with 200 mg/kg/day was considered to be test item-related. However, since there was no histological correlate and no change was observed in kidney or adrenal weights after the recovery period, these differences were considered to be non-adverse.
Decreased brain to body weight ratio in males treated with 50 mg/kg/day was considered to be incidental as it was observed in the low dose group only.
After the recovery period, increased mean absolute liver weight (+14 %, p<0.05) compared to controls was still observed in males treated with 800 mg/kg/day. However, the magnitude of the change was lower than after treatment clearly indicating the tendency to recover.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related macroscopic findings were noted.
The few findings noted in single animals of different groups (discoloration of lungs, constriction of spleen and watery fluid in uterus) were considered to be within the normal range for rats of this strain and age. Beginning autolysis was noted in the control female (no. 38) which died after blood sampling at the end of the recovery period.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At the end of the treatment period, test-item related findings were observed in the liver and potentially in the kidneys of animals treated with 800 mg/kg /day as well as in the kidneys of one male treated with 200 mg/kg/day.
In the liver, minimal centrilobular hepatocellular hypertrophy was recorded in 4/5 males and 2/5 females treated with 800 mg/kg /day with the enlarged hepatocytes displaying a “ground glass appearance”.
In the kidneys, increased hyaline droplets were observed in the renal tubular epithelium of males treated with 800 mg/kg /day (minimal in 2/5 males and slight in 1/5 males) and in one male treated with 200 mg/kg/day (minimal in 1/5 males).
After the recovery period, minimally increased hyaline droplets were still present in the renal tubular epithelium of 2/5 males treated with 800 mg/kg /day.
Slight focal/multifocal congestion of the lungs was the only relevant microscopic finding in female no.38 of the control group, which died spontaneously. The cause of death could not be determined histologically.
All other changes were those commonly seen as spontaneous findings in Wistar rats and had no relationship to treatment with the test item.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Details on results:

ANALYSIS OF DOSE FORMULATIONS
The test item peak was assigned in sample chromatograms by comparison to that of calibration solutions. In blank sample chromatograms no peak appeared at the retention time of the test item and, therefore, the absence of the test item in the vehicle control samples (corn oil) was confirmed.
The test item concentrations in the dose formulations ranged from 96.3% to 117.5% with reference to the nominal and were within the accepted range of ±20%. The homogeneous distribution of the test item in the preparations was approved because single results found did not deviate more than 9.0% from the corresponding mean and met the specified acceptance criterion of ≤15%. In addition, the test item was found to be stable in application formulations when kept four hours at room temperature or eight days in the refrigerator (5 ± 3 °C) due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean value.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Critical effects observed:

no

Dose levels (mg/kg bw/day)	Group 2 - 50 mg/kg bw/day		Group 3 - 200 mg/kg bw/day		Group 4 - 800 mg/kg bw/day
	Males	Females	Males	Females	Males	Females
Liver abs.	+ 13%^#	+ 7%^#	+ 24%**	+ 25%**	+ 24%**	+ 31%**
to bw	+ 4%^#	+ 6%^#	+ 19%**	+ 20%**	+ 22%**	+ 31%**
to brain	+ 16%^#	+ 11%^#	+ 26%**	+ 25%**	+ 28%**	+ 28%**
Adrenales abs.	-	-	-	-	- 24%*	-
to bw	-	-	-	-	- 23%*	-
to brain	-	-	-	-	- 22%*	-
Kidneys abs.	-	-	-	+ 12%^#	-	+ 14%*
to bw	-	-	-	+ 8%^#	-	+ 14%*
to brain	-	-	-	+ 11%*	-	+ 11%*

*: p<0.05; **: p<0.01; #: not significant; Dunnett-test

Conclusions:

Under the conditions of this study and based on effect of the test substance on the animals, the test substance is considered to have a no-observed-adverse-effect-level (NOAEL) in rats of both sexes at 800 mg/kg body weight/day.

Executive summary:

In a repeated dose toxicity study performed in accordance with OECD test guideline No. 407 and in compliance with GLP, test item was administered daily by oral gavage to SPF-bred Wistar rats of both sexes (5/sex) at dose levels of 50, 200 and 800 mg/kg body weight/day for a period of 28 days. A control group was treated similarly with the vehicle, corn oil, only. An additional group of 5 rats per sex dosed at 0 and 800 mg/kg/day and treated for 28 days was allowed a 14-day treatment-free recovery period after which they were sacrificed.

Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during the acclimatization, treatment and recovery periods. Functional observational battery, locomotor activity and grip strength were evaluated during week 4. At the end of the dosing and the treatment-free recovery period, blood samples were withdrawn for hematology and plasma chemistry analyses. Urine samples were collected for urinalyses. All animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues from all control and high dose animals, and all gross lesions from all animals. 

 

No test item-related unscheduled deaths occurred and the spontaneous death of one control female on its scheduled day of necropsy was considered to be related to the blood sampling procedure. Clinical sign of salivation after dosing was noted with all animals at 800 mg/kg/day showing slight to moderate salivation after dosing and/or at later observation on most treatment days, partially with reddish discoloration.
In hematology parameters, slightly increased partial thromboplastin time in males and slightly increased hemoglobin concentration distribution width in females were observed at 800 mg/kg/ay or at 800 and 200 mg/kg/day, respectively. These isolated changes were considered to be possibly test item-related as they were noted in the high dose group but in one sex only. Both changes were still present in males and females treated with 800 mg/kg/day, respectively, at the end of the recovery period. However, due to the low magnitude of the changes and since the values were within the range of historical control data for rats of this strain and age, these changes were considered to be non-adverse.
In clinical chemistry, some statistically significant differences between animals treated with 800 mg/kg/day and control animals at the end of treatment were considered to be possibly test item-related as well because they were noted in the high dose group but generally in one sex only. These changes included e.g. slightly increased glucose and potassium levels and slightly decreased globulin concentration in males, increased creatinine, triglyceride, phospholipid, sodium, phosphorus and albumin concentration in females as well as increased albumin/globulin ratio in males and females. Potassium concentration in males and albumin concentration in females were slightly increased at 200 mg/kg/day as well. No similar changes were present at the end of the recovery period, thus the listed changes were considered not to be adverse.

Decreased mean absolute and relative adrenal weights noted in males treated with 800 mg/kg/day and increased absolute and relative kidney weights noted in females treated with 800 mg/kg/day were considered to be test item-related. However, since there was no histological correlate and no similar change was observed after the recovery period, these differences were considered to be non-adverse.

 

Increased mean absolute and relative liver weights were noted in animals treated with 800 or 200 mg/kg/day. After the recovery period, increased mean absolute liver weight compared to controls was still observed in males treated with 800 mg/kg/day; however, the magnitude of the change was lower than after treatment clearly indicating the tendency to recover. Microscopically, there was minimal centrilobular hepatocellular hypertrophy in both sexes treated with 800 mg/kg/day. These findings are suggestive of an adaptive response to mixed function oxidase induction and not considered adverse.

 

In the kidneys, increased hyaline droplets were observed in the renal tubular epithelium of 2/5 males treated with 800 mg/kg/day and in one male treated with 200 mg/kg/day. After recovery, minimal increase in hyaline droplets were still present in 2/5 males treated with 800 mg/kg/day. The hyaline droplets within the proximal tubules are consistent with the accumulation of alpha-2u-globulin, a common finding in untreated male rats. Although a relationship between the occurrence in males treated with 800 or 200 mg/kg/day and the treatment cannot be excluded, this finding is considered to be specific to the male rat and does not represent a risk to humans. The observed hepatocellular hypertrophy was fully reversible and the incidence and severity of tubular hyaline droplets were reduced after the recovery period therefore both findings were considered not to be adverse.

Based on the results of this study, 800 mg/kg body weight/day of the test item was established as the no-observed-adverse-effect-level (NOAEL) and 50 mg/kg/day as the no-observed-effect-level (NOEL).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

800 mg/kg bw/day

Study duration:

subacute

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

Repeated dose toxicity - oral:

A key sub-chronic toxicity study was identified (Harlan, 2014, rel.1).

In a repeated dose toxicity study performed in accordance with OECD test guideline No. 407 and in compliance with GLP, the test item was administered daily by oral gavage to SPF-bred Wistar rats of both sexes (5/sex) at dose levels of 50, 200 and 800 mg/kg body weight/day for a period of 28 days. A control group was treated similarly with the vehicle, corn oil, only. An additional group of 5 rats per sex dosed at 0 and 800 mg/kg/day and treated for 28 days was allowed a 14-day treatment-free recovery period after which they were sacrificed.

 

Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during the acclimatization, treatment and recovery periods. Functional observational battery, locomotor activity and grip strength were evaluated during week 4. At the end of the dosing and the treatment-free recovery period, blood samples were withdrawn for hematology and plasma chemistry analyses. Urine samples were collected for urinalyses. All animals were euthanized, necropsied and examined post mortem. Histological examinations were performed on organs and tissues from all control and high dose animals, and all gross lesions from all animals. 

 

No test item-related unscheduled deaths occurred and the spontaneous death of one control female on its scheduled day of necropsy was considered to be related to the blood sampling procedure. Clinical sign of salivation after dosing was noted with all animals at 800 mg/kg/day showing slight to moderate salivation after dosing and/or at later observation on most treatment days, partially with reddish discoloration.

 

In hematology parameters, slightly increased partial thromboplastin time in males and slightly increased hemoglobin concentration distribution width in females were observed at 800 mg/kg/day or at 800 and 200 mg/kg/day, respectively. These isolated changes were considered to be possibly test item-related as they were noted in the high dose group but in one sex only. Both changes were still present in males and females treated with 800 mg/kg/day, respectively, at the end of the recovery period. However, due to the low magnitude of the changes and since the values were within the range of historical control data for rats of this strain and age, these changes were considered to be non-adverse. In clinical chemistry, some statistically significant differences between animals treated with 800 mg/kg/day and control animals at the end of treatment were considered to be possibly test item-related as well because they were noted in the high dose group but generally in one sex only. These changes included e.g. slightly increased glucose and potassium levels and slightly decreased globulin concentration in males, increased creatinine, triglyceride, phospholipid, sodium, phosphorus and albumin concentration in females as well as increased albumin/globulin ratio in males and females. Potassium concentration in males and albumin concentration in females were slightly increased at 200 mg/kg/day as well. No similar changes were present at the end of the recovery period, thus the listed changes were considered not to be adverse.

 

Decreased mean absolute and relative adrenal weights noted in males treated with 800 mg/kg/day and increased absolute and relative kidney weights noted in females treated with 800 mg/kg/day were considered to be test item-related. However, since there was no histological correlate and no similar change was observed after the recovery period, these differences were considered to be non-adverse.

 

Increased mean absolute and relative liver weights were noted in animals treated with 800 or 200 mg/kg/day. After the recovery period, increased mean absolute liver weight compared to controls was still observed in males treated with 800 mg/kg/day; however, the magnitude of the change was lower than after treatment clearly indicating the tendency to recover. Microscopically, there was minimal centrilobular hepatocellular hypertrophy in both sexes treated with 800 mg/kg/day. These findings are suggestive of an adaptive response to mixed function oxidase induction and not considered adverse.

 

In the kidneys, increased hyaline droplets were observed in the renal tubular epithelium of 2/5 males treated with 800 mg/kg/day and in one male treated with 200 mg/kg/day. After recovery, minimal increase in hyaline droplets were still present in 2/5 males treated with 800 mg/kg/day. The hyaline droplets within the proximal tubules are consistent with the accumulation of alpha-2u-globulin, a common finding in untreated male rats. Although a relationship between the occurrence in males treated with 800 or 200 mg/kg/day and the treatment cannot be excluded, this finding is considered to be specific to the male rat and does not represent a risk to humans. The observed hepatocellular hypertrophy was fully reversible and the incidence and severity of tubular hyaline droplets were reduced after the recovery period therefore both findings were considered not to be adverse.

 Based on the results of this study, 800 mg/kg body weight/day of the test item was established as the no-observed-adverse-effect-level (NOAEL) and 50 mg/kg/day as the no-observed-effect-level (NOEL).

Justification for classification or non-classification

Harmonised classification:

The test material has no harmonised classification for repeated dose toxicity according to the Regulation (EC) No. 1272/2008 (CLP).

 

Self-classification:

Based on the available information, no additional self-classification is proposed regarding the specific target organ toxicity after oral dose-repeated exposure according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

No information is available regarding the dermal and inhalation route.