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Administrative data

Description of key information

Subacute NOAEL (male/female, rat): 60 mg/kg bw/day (OECD 422/GLP)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
repeated dose toxicity: oral, other
Remarks:
Combined Repeated Dose Toxicity Study with Reproduction/Developmental Toxicity Screening Test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23-10-2017 to 23-11-2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: O’Laughlin (Nantong) Fine Chemicals Co., Ltd.; NTA375
- Expiration date of the lot/batch: Sep 25, 2020
- Purity: CAS No. 67634-00-8: 79.45 %; CAS No.: 67634-01-9 20.28 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Store in cool place. Keep container tightly closed in a dry and well-ventilated place.

Species:
rat
Strain:
other: Wistar CRL
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River SPF breeding, supplied via VELAZ s.r.o., Czech Republic, RČH CZ 11760500
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: males, females: sexually adult, 7 - 9 weeks on arrival ; dose-range finding experiment: 9 weeks on arrival
- Housing: 2 rats of the same sex in one cage in pre-mating period, during mating period – one male and one female in one cage, pregnant females – individually, offspring – with mother, satellite animals - 2 rats of the same sex in one cage. sterilized soft wood fibers Lignocel.
- Diet: maintenance pelleted diet for rats and mice - Altromin for rats/mice ad libitum, Manufacturer: Altromin Spezialfutter GmbH & Co. KG, Germany (Batch No. 151118/0654, exp.15.11.2018)
- Water: drinking water ad libitum
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30-70 %
- Photoperiod (hrs dark / hrs light): 12 hour light / 12 hour dark
Route of administration:
oral: gavage
Vehicle:
olive oil
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
Preparation of the application form was based on the result of the study AAG_467-17-19HS (Annex 1 of this final report). The test item was weighted into a glass beaker and the beaker was replenished by olive oil. The test solution was stirred by magnetic stirrer (500 rpm) for 30 minutes. The concentrations of solution at all dose levels were adjusted to ensure the administration of 1 mL per 100 g of body weight. For each dose level concentration, the solution was prepared separately. The application forms were prepared daily just before administration. The administration of the test item to animals was performed during one hour after preparation of application form. The stirring of solutions continued during administration.

Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
Parental males and satellite animals (males and females): 49 days
Parential females: until the end of the lactation period
Frequency of treatment:
Daily
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
60 mg/kg bw/day (nominal)
Dose / conc.:
120 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Basic groups:
Control: 6 males + 6 females
Low dose: 6 males + 6 females
Intermediate dose: 6 males + 6 females
High dose: 6 males + 6 females

Satellite groups:
Control: 6 males + 6 females
High dose: 6 males + 6 females

Refer to Table 8. The first six males, six mothers who delivered pups per group and a satellite groups of animals (control and treated) are part of the repeated dose toxicity study and examined with respect to toxicity of the test item. Satellite animals were used for observation of reversibility, persistence or delayed occurrence of systemic toxicity effects up to 14 days post treatment.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Due to substantial deaths, two dose-range finding experiments in dose levels 1000, 500, 250, 125 mg/kg/day and 160, 80, 40, 20 mg/kg day had to be performed (Annex 2).

Deaths occurred during the Dose-range finding experiments with the test item Allyl Amyl Glycolate. All males and females at the dose levels 1000, 500 and 250 mg/kg/day died or had to be euthanized due to moribund condition till the third day of experiment. One male died at the dose level 160 mg/kg/day. No death was recorded in animals at the dose level 125, 80, 40 and 20 mg/kg/day.

Clinical symptoms of intoxication as piloerection, hunched posture, apathy, motionless, bradypnoea and decreased reaction to stimuli were recorded in males and females at the dose levels 1000, 500, 250, 125, 160 mg/kg/day.

The lowest body weight of treated animals were recorded in males of the dose levels 125 and 160 mg/kg/day and in females at the dose level 160 mg/kg/day.

The administration of the test item caused slight decrease of total erythrocytes count in males and females at the dose level 160 mg/kg/day compared to other dose levels.

The serious macroscopic changes were observed during necropsy of treated animals. Damage of liver was observed in males and females at the dose levels 1000, 500, 250, 125 and 160 mg/kg/day (males only). Pathological examination of males and females revealed changes of colour and multiple focal changes of liver, enlarged spleen, congested mucosa of small intestine and urinary bladder full of dark red liquid. Males were affected by the test item treatment more than females.

On the basis of the results given above the following dose levels – 120, 60 and 30 mg/kg/day were proposed for the main Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test with the test item Ally Amyl Glycolate.


- Rationale for animal assignment (if not random): The first six males, six mothers with delivered pups per group and satellite groups of animals (control and treated) are a part of the repeated dose toxicity part of study and examined with the respect to toxicity effect of the test item on organism (functional observations, haematology, biochemistry, full biometry and full pathology will be performed in 6 males + 6 females of each dose level and in satellite animals). Satellite animals are used for observation of reversibility, persistence or delayed occurrence of systemic toxicity effect, for 14 days post treatment.


Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes

Health Condition Control
All rats were observed pre-experimentally to ensure that only the animals exhibiting normal behavioural activity would be entered into the study. During the administration period they were examined for behaviour changes each day before application, during application and immediately after application.

Clinical Observations of Parental Males and Females
All rats were observed daily during the administration period. This observation was made in order to record possible clinical effects after application and all changes in behaviour of animals. So it was done after application at the same time every day (12.00 – 14.00 p.m.). Animals were observed in natural conditions in their cages.

Detailed Clinical Parental Observation
This observation was carried out before the first application and then weekly. At the first part of observation the behaviour of animals in the cage was monitored: piloerection, posture, breathing, tonic or clonic movements, stereotypes or bizarre behaviour. The second part was the observation during the removal from cage: reaction to handling, elasticity of skin, colour of mucous membranes, salivation, lacrimation, cleanliness of fur around foramina.

BODY WEIGHT: Yes
- Time schedule for examinations:
Males and satellite animals - the first day of administration and then weekly,
Females - the first day of administration and then weekly,
During pregnancy: 0., 7th, 14th, 20th day,
During lactation: 1st, 4th day, 12th day and 13th day,

FOOD CONSUMPTION AND COMPOUND INTAKE:
Main study: Weekly and on the same days as body weight (except the mating period)
Satellite males and females – weekly

FOOD EFFICIENCY:
-Food conversion in % (weight increment/food consumption x 100) was calculated for animals of Repeated Dose Toxicity part of study.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: The drinking water consumption was recorded in satellite males and females. The mean values in groups (water consumption per animal and per day) were calculated for each week of the study.

HAEMATOLOGY: Yes (Table 3)
Males – after the end of application period
Parental females – on the 13th day of lactation
Satellite animals – after the end of observation period

CLINICAL CHEMISTRY: Yes (Table 4)
Males and nonpregnant females – after the end of application period
Parental females s – on the 13th day of lactation
Satellite animals – after the end of observation period

URINALYSIS: Yes (Table 2)
6 males of each group and in satellite males – 63rd day (main study) and 77th day (satellite group)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of administration/observation period
- Battery of functions tested: Sensory reactivity on auditory, visual, proprioceptive stimuli and pupillary reflex were evaluated and motor activity assessment was conducted. The individual observations of grip strength were performed using a grip strength meter. Measurements were made on: 1) pectoral legs, 2) pelvis legs. Grip power was expressed in Newtons.

Functional observations, haematology and biochemistry were performed only in 6 males + 6 females of each dose level and in satellite animals.

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Pathological examination: males and nonpregnant females – after the end of application period; parental females – on the 13th day of lactation; satellite animals – after the end of observation period
Weight of organs: during necropsy

HISTOPATHOLOGY: Yes (Table 5). Full histopathology of the preserved organs and tissues was performed for all high dose and control animals and satellite animals. Organs with macroscopical changes, liver and spleen were examined at the lowest and middle dose level groups.
Statistics:
Males/females from control group were compared with males/females from three treated groups. Satellite males/females from control group were compared with satellite males/females from treated group. The results statistically significant on probability level (p ≤ 0.05) were indicated in the summary tables.

In general:
The parametric tests were used for statistical evaluation of
•body weight of males and females
•mean pup weight
•litter weight
•anogenital distance of pups
•selected haematology parameters
•blood biochemistry parameters
•data from urinalysis (pH, volume)
•data from biometry of organs

As the first step the test for normality (Shapiro-Wilk test) was used. If the data were not normally distributed than the transformation of data was performed (Box-Cox transformation). If still the normal distributed distribution was not achieved than non-parametric tests (Kruskal-Wallis Test, Mann-Whitney test) were used.

For normally distributed data have at first the variance check has been performed (Levene´s test) to verify if standard deviations within each group are equal. One-Way ANOVA (probability level p ≤ 0.05) was used to detect whether there are any significant differences amongst the means. In case of significant differences, the post hoc statistical testing was performed (Fisher's least significant difference - LSD test).

The non-parametric tests were used for statistical evaluation of
•selected reproduction parameters with non-continuous distribution (number of live born pups, number of pups, number of implantations)
•selected haematology parameters with non-continuous distribution (total erythrocyte count, total leucocyte count, total platelet count)

The Kruskal-Wallis test was used for the comparison of the measured effect in all treatment groups with the vehicle control group (as global test) and the two-groups Mann-Whitney test (probability level p ≤ 0.05).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The health condition control results were noted for all 12 animals per group and satellite animals (Table Nos. 17 - 22).

Males
The behaviour, clinical status and activity of males in the dose groups 30 and 60 mg/kg bw/day were similar during the study and not different from males of the control group. Mild changes were found during examination of hair (piloerection) and salivation was noted in males at the dose level 120 mg/kg bw/day.

Satellite males
Mild changes persisted in satellite treated males at the dose level 120 mg/kg bw/day. During the examination of hair, piloerection was noted and salivation was recorded.

Females
The behaviour, clinical status and activity of females at the dose levels 30 and 60 mg/kg bw/day were similar during the study and not different from females of the control group. Mild changes were found during examination of hair (piloerection) and salivation was noted in females at the dose level 120 mg/kg bw/day.

Satellite females
Mild changes were found in one female at the dose level 120 mg/kg bw/day. During the examination of hair (piloerection), and salivation, excretion (diarrhoea), reaction to handling (decreased reaction to stimuli) were recorded. This response is unlikely to be related to treatment with the test item, because lesions related to improper administration of the test item (intubation error) were noted in the microscopic examination of this female rat No. 193.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled male or female deaths during the repeated dose toxicity part of the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The test item slightly interfered with the normal growth of treated parental females. Body weight of females was significantly decreased during the lactation period at the dose level 120 mg/kg bw/day. Statistically significant differences in necropsy body weight were not found in satellite treated females. Body weight and body weight increment of males were not significantly affected by the test item treatment (Tables 9 – 11).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There were no effects in males (Table 12)

During the pre-mating period, the food consumption of treated females was lower in comparison with control females – except females at the dose level 120 mg/kg bw/day at the end of the 2nd week. During pregnancy, the food consumption of all treated groups of females was slightly lower in comparison with the control group of females. Statistically significantly decreased food consumption was recorded in groups of females at the dose levels 30 and 60 mg/kg bw/day during the pregnancy period. At the end of the lactation period, the food consumptions of females at the dose levels 60 and 120 mg/kg bw/day was markedly decreased (statistically significant) in comparison with the control group of females. This was because one female at the dose level 60 mg/kg bw/day and two females at the dose level 120 mg/kg bw/day lost all their pups (due to death or cannibalism), did not breastfeed and their food consumption was significantly lower. The food consumption of satellite treated females was slightly lower in comparison with the control group during the entire application and recovery periods (Table 13).
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
In males, the food conversion was variable (most of time increased) in the pre-mating period and not negatively influenced by the test item treatment in comparison with the control group of males. During most of the period after mating, the food conversion of males in the treated group was higher (esp. in males at the dose levels 30 and 60 mg/kg bw/day) in comparison with the control males.
The food conversion of satellite treated males was quite similar to the satellite control (exc. the 7th and the 8th week of study).(Table 14)

In females, the food conversion in the pre-mating period was variable but not adversely influenced by the test item treatment. During the pregnancy period, the food conversion of treated females at the dose level 120 mg/kg bw/day was slightly decreased compared to the control group. During the lactation period, the food conversion of treated females at all dose levels was markedly decreased in comparison with the control females. At the end of the lactation period, the food conversion of treated females was similar to the control females. The food conversion of satellite treated females was variable compared to satellite control females, but not adversely influenced by the test item treatment. (Tables 15)

Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
The water consumption of satellite treated males was higher compared to the satellite control group during the entire application period; at the end of the recovery period, the difference was not evident.
The water consumption of satellite treated females was variable in comparison with the satellite control group during the application period (Table 16).
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Haematological examination of males at the dose level 120 mg/kg bw/day revealed statistically significantly changed parameters in comparison with the control males (Table 25): decrease of total erythrocyte count (p ≤ 0.05; outside of historical control range) associated with decreased haemoglobin concentration (p ≤ 0.05; outside of historical control range) and increased value of MCV (p ≤ 0.05; outside of historical control range). There was a dose dependent increase in the percentage value of reticulocytes and there was a statistically significant increase at the dose level 120 mg/kg (outside of the historical control range). The differential leucocyte count was also altered: increased percentage value of granulocytes (p ≤ 0.05; outside of historical control range) and decreased value of monocytes (p ≤ 0.05) were recorded. In satellite treated males, the value of MCV remained statistically significantly increased in comparison with the control satellite animals. The values of haemocoagulation parameters were not significantly affected by the test item treatment, except the value of APTT in males at the dose level 30 mg/kg bw/day.

Haematological examination of females demonstrated statistically significantly changed parameters only in females at the dose level 120 mg/kg bw/day in comparison with the control females (Tables 26). A decrease in the total erythrocyte count (p ≤ 0.05; outside of historical control range) associated with decreased haemoglobin concentration (p ≤ 0.05) and value of haematocrite (p ≤ 0.05) were found. An increased in the value of MCV (without statistical significance but outside of historical control range) was also recorded. The percentage value of reticulocytes was increased (outside of historical control range) in females at the dose level 120 mg/kg bw/day. At 120 mg/kg bw/day, the value of total leucocyte count (WBC) was increased statistically significantly. The differential leucocyte count was changed as follows: decreased percentage value of granulocytes in females at all dose levels (dose dependent), increased values of lymphocytes and monocytes in females at all dose levels. Haematocoagulation parameter were changed as follows: decreased value of APTT (p ≤ 0.05), increased value of PT (p ≤ 0.05; outside of historical control range) in females at the dose level 120 mg/kg bw/day. In satellite treated females, some of the parameters were changed statistically insignificantly, but parameters were outside of the historical control range in comparison with the control satellite animals. The RBC value recovered to a similar level compared to control animals. An increased value (outside of historical control range) of MCV, granulocytes and reticulocytes were recorded in satellite treated females.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):

During biochemical examination of males, statistically significantly changed values (compared to control) in liver enzyme concentrations were recorded (Table 27). In males at the dose level 120 mg/kg bw/day, statistically significantly increased concentrations of liver enzymes – ALP (outside of historical control range), ALT (outside of historical control range) and AST (insignificantly, but outside of historical control range) were recorded. The mean value of T-Bil was increased at 120 mg/kg bw/day (outside of historical control range). In males at the dose level 60 mg/kg bw/day, the value of ALP was also statistically significantly increased, the value of ALT was increased insignificantly. The concentration of potassium ions was increased significantly (outside of historical control range). In males at the dose level 30 mg/kg, the values of ALP and ALT were insignificantly increased and the values of ALB and GLU were increased statistically significantly. In satellite treated males increased value of creatinine was recorded.

During the biochemical examination of females, statistically significantly changed values (compared to control) in liver enzyme concentration were recorded (Table 28). In females at the dose level 120 mg/kg bw/day, there was an increase in ALP (p ≤ 0.05; outside of historical control range), ALT (insignificantly, outside of historical control range) and the mean value of T-Bil was significantly increased (outside of historical control range). Statistically significant decreases in Crea, BUN (out of historical control range) and GLU were recorded. In females at the dose level 60 mg/kg bw/day, the value of ALP and T-Bil were insignificantly increased. In females at the dose level 30 mg/kg, the values of ALP and ALT (outside of historical control range) were insignificantly increased In satellite treated females, decrease in the values of Ca and T-Bil (p ≤ 0.05) was recorded.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
An increased volume of urine (p ≤ 0.05) was detected in males at the dose level 60 mg/kg bw/day (Table 29). The presence of proteins, blood and leucocytes were recorded in treated males as well as in control males and were not associated with the application of the test item.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No differences in functional observations were noted in any males/females throughout the study (Tables 23-24).
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Biometry of organs of treated animals showed significant changes in weight of some organs in comparison with the control animals. In males at the dose level 120 mg/kg bw/day, there was an increase in the absolute weight of spleen and the relative weight of spleen was increased statistically significantly (Tables 30, 32). Absolute and relative weights of the kidneys and liver were statistically significantly increased. In males at the dose level 30 mg/kg bw/day, the absolute and relative weight of spleen was significantly decreased. In satellite treated males, a statistically significant increase in the absolute and relative weight of the prostate gland with seminal vesicles was recorded.

In females at the dose level 120 mg/kg bw/day, a statistically significant increase in the absolute and relative weight of the spleen and a decrease in the absolute weight of the brain was recorded (Tables 31, 33). In females at the dose level 60 mg/kg bw/day, the absolute weight of the brain and adrenal glands was significantly decreased. The absolute weight of the adrenal glands was significantly decreased in females at the dose level 30 mg/kg bw/day. Statistically significant differences were not recorded in the absolute or relative weights of organs in satellite females.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Macroscopical findings in the liver and spleen were diagnosed in animals mainly at the dose level 120 mg/kg bw/day during the pathological examination. Multiple focal changes of light colour on the liver, altered liver appearance, enlarged spleen were recorded in males and females. Sporadic findings were diagnosed in animals at the dose level 60 mg/kg bw/day. No macroscopical findings were recorded in animals at the dose level 30 mg/kg bw/day.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histological examination recorded changes related to the test item treatment in males and females (Tables 34, 35). Focal necrosis of differing severity and mostly of periportal localization were found in the liver parenchyma of 4/6 males and 4/6 females from the high dose group. Further, minimal to mild bile duct hyperplasia was observed in the liver of all 6 males and 6 females. The presence of small amounts of brown pigment (possibly hemosiderin) was noted in the liver of 1/6 males and 4/6 females. These lesions were in direct relation to the test item administered. Recovered high dose rats showed minimal bile duct hyperplasia in the liver of 4/6 males and 2/6 females, together with the presence of a small amount of brown pigment in 2/6 and 2/6 females and a small solitary granuloma in 1/6 male and 2/6 females. Minimal periportal fibrosis was seen in the liver of 2/6 recovered males.
Histopathological findings: neoplastic:
no effects observed
Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
clinical biochemistry
gross pathology
histopathology: non-neoplastic
Critical effects observed:
yes
Lowest effective dose / conc.:
60 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
In a combined repeated dose and reproduction/developmental toxicity screening test in Wistar rats, the NOAEL for repeated dose toxcity in males and females was established as 60 mg/kg body weight/day. This judgement is based predominantly on changes of biochemical parameters (liver enzymes), macroscopical and microscopical findings on liver and spleen in animals at the dose level 120 mg/kg bw/day. The NOEL for repeated dose toxcity in males and females was established as 30 mg/kg body weight/day.
Executive summary:

In a combined repeated dose and reproduction/developmental toxicity screening test (18-343), Allyl Amyl Glycolate was administered to 6 groups of Wistar rats (6 animals/sex/group) by gavage in olive oil at dose levels of 0, 30, 60 and 120 mg/kg bw/day (main study; 0 and 120 mg/kg bw/day for satellite animals), 7 days per week, for 49 days (parental males and all satellite animals) and until the end of the lactation period (parental females). Satellite animals were used for observation of reversibility, persistence or delayed occurrence of systemic toxicity effects for 14 days post treatment.

There were no unscheduled male or female deaths during the repeated dose toxicity part of the study. Test item treatment produced clinical changes such as piloerection, salivation, apathy and decreased reaction to stimuli after application in males and females. The test item slightly interfered with the normal growth of treated parental females. Body weight of females was significantly decreased during the lactation period at the dose level 120 mg/kg bw/day. Statistically significant differences in necropsy body weight were not found in satellite treated females. Body weight and body weight increment of males were not significantly affected by the test item treatment.  No differences in functional observations were noted in any males/females throughout the study.

Haematological examination of males at the dose level 120 mg/kg bw/day revealed statistically significantly changed parameters in comparison with the control males: decrease of total erythrocyte count (p ≤ 0.05; outside of historical control range) associated with decreased haemoglobin concentration (p ≤ 0.05; outside of historical control range) and increased value of MCV (p ≤ 0.05; outside of historical control range). There was a dose dependent increase in the percentage value of reticulocytes and there was a statistically significant increase at the dose level 120 mg/kg (outside of the historical control range). The differential leucocyte count was also altered: increased percentage value of granulocytes (p ≤ 0.05; outside of historical control range) and decreased value of monocytes (p ≤ 0.05) were recorded. In satellite treated males, the value of MCV remained statistically significantly increased in comparison with the control satellite animals. The values of haemocoagulation parameters were not significantly affected by the test item treatment, except the value of APTT in males at the dose level 30 mg/kg bw/day. Haematological examination of females demonstrated statistically significantly changed parameters only in females at the dose level 120 mg/kg bw/day in comparison with the control females. A decrease in the total erythrocyte count (p ≤ 0.05; outside of historical control range) associated with decreased haemoglobin concentration (p ≤ 0.05) and value of haematocrite (p ≤ 0.05) were found. An increased in the value of MCV (without statistical significance but outside of historical control range) was also recorded. The percentage value of reticulocytes was increased (outside of historical control range) in females at the dose level 120 mg/kg bw/day. At 120 mg/kg bw/day, the value of total leucocyte count (WBC) was increased statistically significantly. The differential leucocyte count was changed as follows: decreased percentage value of granulocytes in females at all dose levels (dose dependent), increased values of lymphocytes and monocytes in females at all dose levels. Haematocoagulation parameter were changed as follows: decreased value of APTT (p ≤ 0.05), increased value of PT (p ≤ 0.05; outside of historical control range) in females at the dose level 120 mg/kg bw/day. In satellite treated females, some of the parameters were changed statistically insignificantly, but parameters were outside of the historical control range in comparison with the control satellite animals. The RBC value recovered to a similar level compared to control animals. An increased value (outside of historical control range) of MCV, granulocytes and reticulocytes were recorded in satellite treated females.

During biochemical examination of males, statistically significantly changed values (compared to control) in liver enzyme concentrations were recorded.  In males at the dose level 120 mg/kg bw/day, statistically significantly increased concentrations of liver enzymes – ALP (outside of historical control range), ALT (outside of historical control range) and AST (insignificantly, but outside of historical control range) were recorded.  The mean value of T-Bil was increased at 120 mg/kg bw/day (outside of historical control range). In males at the dose level 60 mg/kg bw/day, the value of ALP was also statistically significantly increased, the value of ALT was increased insignificantly. The concentration of potassium ions was increased significantly (outside of historical control range). In males at the dose level 30 mg/kg, the values of ALP and ALT were insignificantly increased and the values of ALB and GLU were increased statistically significantly. In satellite treated males, an increased value of creatinine was recorded. During the biochemical examination of females, statistically significantly changed values (compared to control) in liver enzyme concentration were recorded.  In females at the dose level 120 mg/kg bw/day, there was an increase in ALP (p ≤ 0.05; outside of historical control range), ALT (insignificantly, outside of historical control range) and the mean value of T-Bil was significantly increased (outside of historical control range). Statistically significant decreases in Crea, BUN (out of historical control range) and GLU were recorded. In females at the dose level 60 mg/kg bw/day, the value of ALP and T-Bil were insignificantly increased. In females at the dose level 30 mg/kg, the values of ALP and ALT (outside of historical control range) were insignificantly increased In satellite treated females, decrease in the values of Ca and T-Bil (p ≤ 0.05) was recorded.

An increased volume of urine (p ≤ 0.05) was detected in males at the dose level 60 mg/kg bw/day. The presence of proteins, blood and leucocytes were recorded in treated males as well as in control males and were not associated with the application of the test item.

In males at the dose level 120 mg/kg bw/day, there was an increase in the absolute weight of spleen and the relative weight of spleen was increased statistically significantly. Absolute and relative weights of the kidneys and liver were statistically significantly increased. In males at the dose level 30 mg/kg bw/day, the absolute and relative weight of spleen was significantly decreased. In satellite treated males, a statistically significant increase in the absolute and relative weight of the prostate gland with seminal vesicles was recorded. In females at the dose level 120 mg/kg bw/day, a statistically significant increase in the absolute and relative weight of the spleen and a decrease in the absolute weight of the brain was recorded. In females at the dose level 60 mg/kg bw/day, the absolute weight of the brain and adrenal glands was significantly decreased. The absolute weight of the adrenal glands was significantly decreased in females at the dose level 30 mg/kg bw/day. Statistically significant differences were not recorded in the absolute or relative weights of organs in satellite females.

Macroscopical findings in the liver and spleen were diagnosed in animals mainly at the dose level 120 mg/kg bw/day during the pathological examination. Multiple focal changes of light colour on the liver, altered liver appearance, enlarged spleen were recorded in males and females. Sporadic findings were diagnosed in animals at the dose level 60 mg/kg bw/day. No macroscopical findings were recorded in animals at the dose level 30 mg/kg bw/day.

Histological examination recorded changes related to the test item treatment in males and females. Focal necrosis of differing severity and mostly of periportal localization were found in the liver parenchyma of 4/6 males and 4/6 females from the high dose group. Further, minimal to mild bile duct hyperplasia was observed in the liver of all 6 males and 6 females. The presence of small amounts of brown pigment (possibly hemosiderin) was noted in the liver of 1/6 males and 4/6 females. These lesions were in direct relation to the test item administered.  Recovered high dose rats showed minimal bile duct hyperplasia in the liver of 4/6 males and 2/6 females, together with the presence of a small amount of brown pigment in 2/6 and 2/6 females and a small solitary granuloma in 1/6 male and 2/6 females. Minimal periportal fibrosis was seen in the liver of 2/6 recovered males.

Bile duct proliferation participates in the repair of liver damage. Proliferated bile ducts extend into the area of necrosis during the first week following periportal necrosis of hepatocytes. In the latter instance, the periportal area may be repopulated by a mixture of hepatocytes and normal-appearing bile ducts. Minimal fibrosis may also extend from the portal area in the reparative process. The test item has a direct hepatotoxic effect (liver necrosis), so the liver is a target organ. Bile duct proliferation observed is considered to be a part of reparative process, so its relation to the test item is most probably indirect. Subsequent histopathological examination of the liver of the middle and low dose rats found hemorrhagic necrosis in 1/6 males at the middle dose level and the presence of a minimal amount of brown pigment in the liver of 1/6 middle dose female.  

Mild to marked extramedullary hemopoiesis was found in the spleen of 3/6 males and 3/6 females from the high dose group and one recovered high dose female in comparison to its minimal grade found in one control male. This finding was most probably related to the test item administration and led to subsequent examination of the spleen in the middle and low dose groups of rats. Extramedullary hemopoiesis (mild grade) was found in the spleen of one middle dose male and of minimal grade in one low dose female.

The NOAEL (No Observed Adverse Effect Level) for repeated dose toxcity in males and females was established as 60 mg/kg body weight/day. This judgement is based predominantly on changes of biochemical parameters (liver enzymes), macroscopical and microscopical findings on liver and spleen in animals at the dose level 120 mg/kg bw/day. The NOEL (No Observed Effect Level) for repeated dose toxcity in males and females was established as 30 mg/kg body weight/day.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
60 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The key study was an OECD 422/GLP study and is the only study available. It was assigned a Klimisch score of 1.
System:
hepatobiliary
Organ:
liver

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

There is one combined repeated dose and reproduction/developmental toxicity screening test in rats available.

In a combined repeated dose and reproduction/developmental toxicity screening test (OECD 422/GLP), Allyl Amyl Glycolate was administered to 6 groups of Wistar rats (6 animals/sex/group) by gavage in olive oil at dose levels of 0, 30, 60 and 120 mg/kg bw/day (main study; 0 and 120 mg/kg bw/day for satellite animals), 7 days per week, for 49 days (parental males and all satellite animals) and until the end of the lactation period (parental females). Satellite animals were used for observation of reversibility, persistence or delayed occurrence of systemic toxicity effects for 14 days post treatment.

There were no unscheduled male or female deaths during the repeated dose toxicity part of the study. Test item treatment produced clinical changes such as piloerection, salivation, apathy and decreased reaction to stimuli after application in males and females. The test item slightly interfered with the normal growth of treated parental females. Body weight of females was significantly decreased during the lactation period at the dose level 120 mg/kg bw/day. Statistically significant differences in necropsy body weight were not found in satellite treated females. Body weight and body weight increment of males were not significantly affected by the test item treatment.  No differences in functional observations were noted in any males/females throughout the study.

Haematological examination of males at the dose level 120 mg/kg bw/day revealed statistically significantly changed parameters in comparison with the control males: decrease of total erythrocyte count (p ≤ 0.05; outside of historical control range) associated with decreased haemoglobin concentration (p ≤ 0.05; outside of historical control range) and increased value of MCV (p ≤ 0.05; outside of historical control range). There was a dose dependent increase in the percentage value of reticulocytes and there was a statistically significant increase at the dose level 120 mg/kg (outside of the historical control range). The differential leucocyte count was also altered: increased percentage value of granulocytes (p ≤ 0.05; outside of historical control range) and decreased value of monocytes (p ≤ 0.05) were recorded. In satellite treated males, the value of MCV remained statistically significantly increased in comparison with the control satellite animals. The values of haemocoagulation parameters were not significantly affected by the test item treatment, except the value of APTT in males at the dose level 30 mg/kg bw/day. Haematological examination of females demonstrated statistically significantly changed parameters only in females at the dose level 120 mg/kg bw/day in comparison with the control females. A decrease in the total erythrocyte count (p ≤ 0.05; outside of historical control range) associated with decreased haemoglobin concentration (p ≤ 0.05) and value of haematocrite (p ≤ 0.05) were found. An increased in the value of MCV (without statistical significance but outside of historical control range) was also recorded. The percentage value of reticulocytes was increased (outside of historical control range) in females at the dose level 120 mg/kg bw/day. At 120 mg/kg bw/day, the value of total leucocyte count (WBC) was increased statistically significantly. The differential leucocyte count was changed as follows: decreased percentage value of granulocytes in females at all dose levels (dose dependent), increased values of lymphocytes and monocytes in females at all dose levels. Haematocoagulation parameter were changed as follows: decreased value of APTT (p ≤ 0.05), increased value of PT (p ≤ 0.05; outside of historical control range) in females at the dose level 120 mg/kg bw/day. In satellite treated females, some of the parameters were changed statistically insignificantly, but parameters were outside of the historical control range in comparison with the control satellite animals. The RBC value recovered to a similar level compared to control animals. An increased value (outside of historical control range) of MCV, granulocytes and reticulocytes were recorded in satellite treated females.

During biochemical examination of males, statistically significantly changed values (compared to control) in liver enzyme concentrations were recorded.  In males at the dose level 120 mg/kg bw/day, statistically significantly increased concentrations of liver enzymes – ALP (outside of historical control range), ALT (outside of historical control range) and AST (insignificantly, but outside of historical control range) were recorded.  The mean value of T-Bil was increased at 120 mg/kg bw/day (outside of historical control range). In males at the dose level 60 mg/kg bw/day, the value of ALP was also statistically significantly increased, the value of ALT was increased insignificantly. The concentration of potassium ions was increased significantly (outside of historical control range). In males at the dose level 30 mg/kg, the values of ALP and ALT were insignificantly increased and the values of ALB and GLU were increased statistically significantly. In satellite treated males, an increased value of creatinine was recorded. During the biochemical examination of females, statistically significantly changed values (compared to control) in liver enzyme concentration were recorded.  In females at the dose level 120 mg/kg bw/day, there was an increase in ALP (p ≤ 0.05; outside of historical control range), ALT (insignificantly, outside of historical control range) and the mean value of T-Bil was significantly increased (outside of historical control range). Statistically significant decreases in Crea, BUN (out of historical control range) and GLU were recorded. In females at the dose level 60 mg/kg bw/day, the value of ALP and T-Bil were insignificantly increased. In females at the dose level 30 mg/kg, the values of ALP and ALT (outside of historical control range) were insignificantly increased In satellite treated females, decrease in the values of Ca and T-Bil (p ≤ 0.05) was recorded.

An increased volume of urine (p ≤ 0.05) was detected in males at the dose level 60 mg/kg bw/day. The presence of proteins, blood and leucocytes were recorded in treated males as well as in control males and were not associated with the application of the test item.

In males at the dose level 120 mg/kg bw/day, there was an increase in the absolute weight of spleen and the relative weight of spleen was increased statistically significantly. Absolute and relative weights of the kidneys and liver were statistically significantly increased. In males at the dose level 30 mg/kg bw/day, the absolute and relative weight of spleen was significantly decreased. In satellite treated males, a statistically significant increase in the absolute and relative weight of the prostate gland with seminal vesicles was recorded. In females at the dose level 120 mg/kg bw/day, a statistically significant increase in the absolute and relative weight of the spleen and a decrease in the absolute weight of the brain was recorded. In females at the dose level 60 mg/kg bw/day, the absolute weight of the brain and adrenal glands was significantly decreased. The absolute weight of the adrenal glands was significantly decreased in females at the dose level 30 mg/kg bw/day. Statistically significant differences were not recorded in the absolute or relative weights of organs in satellite females.

Macroscopical findings in the liver and spleen were diagnosed in animals mainly at the dose level 120 mg/kg bw/day during the pathological examination. Multiple focal changes of light colour on the liver, altered liver appearance, enlarged spleen were recorded in males and females. Sporadic findings were diagnosed in animals at the dose level 60 mg/kg bw/day. No macroscopical findings were recorded in animals at the dose level 30 mg/kg bw/day.

Histological examination recorded changes related to the test item treatment in males and females. Focal necrosis of differing severity and mostly of periportal localization were found in the liver parenchyma of 4/6 males and 4/6 females from the high dose group. Further, minimal to mild bile duct hyperplasia was observed in the liver of all 6 males and 6 females. The presence of small amounts of brown pigment (possibly hemosiderin) was noted in the liver of 1/6 males and 4/6 females. These lesions were in direct relation to the test item administered.  Recovered high dose rats showed minimal bile duct hyperplasia in the liver of 4/6 males and 2/6 females, together with the presence of a small amount of brown pigment in 2/6 and 2/6 females and a small solitary granuloma in 1/6 male and 2/6 females. Minimal periportal fibrosis was seen in the liver of 2/6 recovered males.

Bile duct proliferation participates in the repair of liver damage. Proliferated bile ducts extend into the area of necrosis during the first week following periportal necrosis of hepatocytes. In the latter instance, the periportal area may be repopulated by a mixture of hepatocytes and normal-appearing bile ducts. Minimal fibrosis may also extend from the portal area in the reparative process. The test item has a direct hepatotoxic effect (liver necrosis), so the liver is a target organ. Bile duct proliferation observed is considered to be a part of reparative process, so its relation to the test item is most probably indirect. Subsequent histopathological examination of the liver of the middle and low dose rats found hemorrhagic necrosis in 1/6 males at the middle dose level and the presence of a minimal amount of brown pigment in the liver of 1/6 middle dose female.  

Mild to marked extramedullary hemopoiesis was found in the spleen of 3/6 males and 3/6 females from the high dose group and one recovered high dose female in comparison to its minimal grade found in one control male. This finding was most probably related to the test item administration and led to subsequent examination of the spleen in the middle and low dose groups of rats. Extramedullary hemopoiesis (mild grade) was found in the spleen of one middle dose male and of minimal grade in one low dose female.

The NOAEL (No Observed Adverse Effect Level) for repeated dose toxcity in males and females was established as 60 mg/kg body weight/day. This judgement is based predominantly on changes of biochemical parameters (liver enzymes), macroscopical and microscopical findings on liver and spleen in animals at the dose level 120 mg/kg bw/day. The NOEL (No Observed Effect Level) for repeated dose toxcity in males and females was established as 30 mg/kg body weight/day.

Justification for classification or non-classification

Based on the available information in the dossier, the substance Allyl Amyl Glycolate (EC No. 916-328-0) is classified for specific target organ toxicity category 2 (repeated; oral (liver)) when the criteria outlined in Annex I of 1272/2008/EC are applied.