Allyl phenoxyacetate

Endpoint summary

Description of key information

Oral (OECD 422), rat: NOAEL 50 mg/kg bw/day in males and females

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 Feb - 08 Aug 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 29, 2016

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD (SD), SPF

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 10 weeks (males); 12 weeks (females)
- Weight at study initiation: 355.0 - 439.4 g (males); 242.3 - 295.3 g (females)
- Housing: During acclimation, pre-treatment, treatment, postmating and recovery period: 2 animals per cage; Mating period: 1 male and 1 female; Gestation period: 1 mated female; Lactation period: neonates were kept with the dam; animals were kept in stainless-steel cages (255W×465L×200H mm) and dams with pups in polycarbonate cages (260W x 420D x 180H mm) on aspen animal bedding
- Diet: Lab Diet® #5053 (PMI Nutrition International, USA) irradiated by gamma-ray, ad libitum
- Water: filtered, ultraviolet light-irradiated municipal tap water, ad libitum
- Acclimation period: 6 days

DETAILS OF FOOD AND WATER QUALITY: Microbial monitoring for diet was performed and a certificate of analysis for the diet was provided by the supplier. The drinking water was analyzed every 6 months for specified contaminants.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 - 22.8
- Humidity (%): 40.9 - 53.8
- Air changes (per hr): 10 - 20
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of the test item was weighed and suspended in corn oil with a magnetic stirrer for about 5 min to prepare the target concentration. The high dose formulation was prepared first and then the lower dose formulations were prepared by diluting the higher dose formulation with corn oil. Dose formulations were prepared at least one time per week and stored at room temperature. Dose formulations were transferred to the animal room at room temperature.

VEHICLE
- Justification for use and choice of vehicle: Corn oil was considered non-toxic with this dose volume (2 mL/kg), and it has been used in previous studies because of the solubility of the test item with this vehicle.
- Amount of vehicle: 2 mL/kg
- Lot/batch no.: MKBZ9899V

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The verification of the dose level concentration was determined by gaschromatography prior to dosing. Samples were taken from the top, middle and bottom of all dose formulations. Results of dose formulations were 106.84, 102.04, and 101.53% at the dose levels of 7.5, 25, and 75 mg/mL, respectively. They were acceptable as the mean concentration was within ±15% of the nominal concentration.

Duration of treatment / exposure:

Main groups:
males: at least 50 days, starting 2 weeks before mating, during mating until the day prior to sacrifice
females: for 2 weeks prior to mating and continued until lactation day 13

Recovery groups:
at least 50 days; Animals were not mated and were assigned to 2 weeks of recovery period after the completion of administration.

Frequency of treatment:

once daily, 7 days/week

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

12 (main groups)
6 (recovery groups; for control and high dose groups)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose level was selected based on the results of a repeated 2-week dose range-finding study in Sprague-Dawley rats in which doses of 15, 60 and 240/150 mg/kg bw/day have been tested. In that study, one found dead female was observed on treatment day 5 at dose level of 240 mg/kg bw/day. The dose level of the high dose group was changed from 240 to 150 mg/kg bw/day on treatment day 6. Increased AST, ALT and yellow discoloration of liver were observed in males at 60 mg/kg bw/day. At 240/150 mg/kg bw/day, general toxicity including soft feces, lacrimation and soiled fur in males, and decreased body weight gain and food consumption in both sexes were observed. In addition, an increased AST and ALT were observed in males. Absolute and relative spleen weights were increased in both sexes. Based on the result of this dose range-finding study, 150 mg/kg bw/day was selected as the high dose, and 50 and 15 mg/kg bw/day were selected as the middle and the low dose, respectively. Animals of the vehicle control were administered with the vehicle alone (corn oil).

- Rationale for animal assignment: Animals were selected for use in the study on the basis of adequate body weight (recorded on the day of receipt and of group assignment), estrus cycle and for being free from clinical signs of disease or injuries during the acclimation and pre-treatment periods. They were randomized and assigned to treatment groups to have a similar mean body weight distribution using the Pristima system based on the most recent body weight.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality and morbidity observations were conducted twice daily except for the acclimation and recovery period where animals were observed once daily. In addition, it was conducted once on necropsy day.
- Cage side observations included: mortality/viability, clinical signs and general condition; during the gestation period: signs of abortion or premature delivery

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once per week
- Observations included: evaluation of fur, skin, eyes, mucous membranes, occurrence of secretions and excretions, autonomic nervous system activity (lacrimation, piloerection, pupil size, and unusual respiratory pattern), changes in gait, posture, clonic and tonic movements, stereotypical and bizarre behavior, and difficult and prolonged parturition

BODY WEIGHT: Yes
- Time schedule for examinations: first dosing day and then once per week; additionally on gestation days 0, 7, 14 and on lactation days 0, 4 and 13

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption per animal [g food/animal/day] was calculated by subtracting the amount of residual feed from the amount presented

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HEMATOLOGY: Yes
- Time schedule for collection of blood: day of scheduled sacrifice
- Anaesthetic used for blood collection: not specified
- Animals fasted: 16 h (overnight) prior to scheduled sacrifice
- How many animals: all scheduled sacrifice animals in the main and the recovery groups
- Parameters checked: total leukocyte count (WBC), mean corpuscular hemoglobin (MCH), total red blood cell count (RBC), mean corpuscular hemoglobin concentration (MCHC), hemoglobin (HGB), platelet count (PLT), hematocrit (HCT), reticulocyte count (absolute (RETA) and relative (RET%) counts), mean corpuscular volume (MCV), WBC differential count (absolute and relative (%) differential counts: including neutrophils (NEU), eosinophils (EOS), basophils (BAS), monocytes (MON), lymphocytes (LYM) and other cells as appropriate (i.e.; large unstained cells (LUC)), prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day of scheduled sacrifice
- Animals fasted: 16 h (overnight) prior to scheduled sacrifice
- How many animals: all scheduled sacrifice animals in the main and the recovery groups
- Parameters checked: glucose (GLU), alanine aminotransferase (ALT), blood urea nitrogen (BUN), total bilirubin (TBIL), creatinine (CREA), alkaline phosphatase (ALP), total protein (TP), gamma glutamyl transpeptidase (GGT), albumin (ALB), creatine phosphokinase (CK), albumin/globulin ratio (A/G), calcium (Ca), total cholesterol (TCHO), inorganic phosphorus (IP), triglyceride (TG), sodium (Na), phospholipid (PL), potassium (K), aspartate aminotransferase (AST), chloride (Cl)

URINALYSIS:No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: shortly before scheduled sacrifice
- Dose groups that were examined: first six animals per sex in main group (if possible) and in all animals of the recovery group
- Battery of functions tested: sensory function tests (approach and touch response, tail pinch, acoustic startle response and pupillary reflex), grip strength and motor activity

IMMUNOLOGY: No

OTHER:
Thyroid Hormone Analysis:
- Time schedule for examinations: at termination
- Dose groups that were examined: all adult animals in main and recovery group
- Parameters checked: thyroid hormone (T4) and the thyroid stimulating hormone (TSH)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The animals were carefully examined for external abnormalities. The abdominal, thoracic and cranial cavities were examined for abnormalities and the organs were removed and examined. Special attention was paid to the organs of the reproductive system. For lactating dams, the number of implantation sites (right and left) was counted. For non-parturition females, uterus implantation sites were examined after staining with ammonium sulfide according to KIT SOPs. Pregnancy was confirmed by implantation sites in the uterus.

ORGAN WEIGHTS: Yes (see table 1)
Paired organs were weighed together unless gross abnormalities were present. However, paired reproductive organs were weighed separately. Animals were fasted overnight prior to necropsy and body weights were recorded on the day of necropsy. Organs were weighed and organ/body weight ratios were calculated.

HISTOPATHOLOGY: Yes (see table 2)
Tissues shown in table 2 from each animal were preserved in 10% neutral buffered formalin, except the eyes (with optic nerve) which were fixed in Davidson’s fixative, and the testes and epididymides which were fixed in Bouin’s fixative. The tissues were placed in the appropriate fixative for approximately 48 hours, and then transferred to 70% ethanol. Formalin was infused into the lung via the trachea and into the urinary bladder.
All tissues except reproductive organs collected from first six animals per sex in main group were further processed to slides, stained with hematoxylin and eosin (H&E), and examined microscopically. All reproductive organs collected from the main groups were further processed to slides, stained with H&E, and examined microscopically. Target organs from the recovery group animals were additionally evaluated microscopically.

Statistics:

Mean values and standard deviations were calculated. Statistical analyses for comparisons of the various dose groups with the vehicle control group were conducted using the Pristima System (Version 7.X. Xybion Medical Systems Corporation, USA) or Statistical Analysis Systems (SAS/STAT Version 9.2 and 9.4, USA). Data were considered to be significant when p<0.05 or p<0.01.
Multiple comparison tests for different dose groups were conducted. Variance of homogeneity was examined using the Bartlett’s Test. Homogeneous data were analyzed using the Analysis of Variance (ANOVA), and the significance of inter-group differences were analyzed using the Dunnett’s Test. Heterogeneous data were analyzed using the Kruskal-Wallis Test, and the significance of inter-group differences was assessed between the control and treated groups using the Dunn’s Rank Sum Test.
For comparing the control group with the recovery group, the data were analyzed for homogeneity for variance using the F-test. Homogeneous data were analyzed using the T-test and the significant difference was assessed between control and recovery group using the Dunnett’s Test. Heterogeneous data were analyzed using the Kruskal-Wallis Test and significant difference was assessed between control and recovery group using the Dunn’s Rank Sum Test.
Data presented as frequencies were analyzed by χ2-test followed by the Fisher's exact test where necessary.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related moribund animals were observed in any group throughout the study.
In males at 15, 50 and 150 mg/kg bw/day, salivation was observed in 11, 12, and 18 animals, respectively. In females at 15, 50, and 150 mg/kg bw/day, salivation was observed in 6, 12, and 18 animals, respectively. It was considered test item-related but not toxicologically significant since it was considered to be attributed to the palatability of the test item.
Other clinical signs were observed in this study but were not considered test item-related since these findings were observed with low frequency or occurred sporadically and did not have any dose-response.

In one female of control group found dead on gestation day 21 one early resorption, one late resorption and 7 dead fetuses were observed in the uterine examination. In the result of microscopic findings, moderate hemorrhage, slight alveolar edema and moderate perivascular congestion were observed in the lung. Moderate centrilobular necrosis in liver, right atrium dilatation in heart, slight atrophy in spleen and minimal mononuclear cell infiltration and hemorrhage in uterus were also observed. These were considered to be incidental since it was observed in the control group and there were no correlated microscopic changes in other animals in the same control group.

Non-parturition was observed in two females of control group and in one female at 150 mg/kg bw/day. Three females were subjected to unscheduled sacrifice on gestation day 27 and all of them were non-pregnant. In the results of microscopic findings, minimal squamous cell hyperplasia in stomach was observed in one animal at 150 mg/kg bw/day, but there were no changes observed in the reproductive system in this animal.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No test item-related deaths occurred in any group throughout the study.
One female of control group was found dead on gestation day (GD) 21. Two females of control group and one female at 150 mg/kg bw/day were subjected to unscheduled sacrifice on gestation day 27 since they were non-pregnant.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test item-related changes in body weight and in body weight gain were observed in both sexes during the study. Statistically significant changes in body weight gain during the study were not considered test item-related since they were transient.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No test item-related changes in food consumption were observed in both sexes during the study.
A statistically significant change in food consumption during the study was not considered test item-related since there was no correlation with body weight changes and there was no dose-dependency.

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):

In males, total leukocyte count (WBC, 1.40-fold over control), absolute lymphocyte count (LYMA, 1.47-fold over control), absolute monocyte count (MONA, 1.43-fold over control), absolute large unstained cells counts (LUCA, 1.33-fold over control), fibrinogen (FIB, 1.22-fold over control) were increased at 150 mg/kg bw/day and platelet count (PLT, up to 1.21-folds over control) was increased at 50 and 150 mg/kg bw/day. The increases of WBC, LYMA, FIB and PLT were statistically significant. In females, WBC (1.30-fold over control), LYMA (1.37-fold over control), MONA (1.62-fold over control) and LUCA (1.58-fold over control) were increased at 150 mg/kg bw/day. The increases of WBC, MONA and LUCA were statistically significant. These changes recovered or showed a tendency of recovery at the end of the recovery period. Increased leukocyte counts, fibrinogen and platelet count in males and/or females at 150 mg/kg bw/day were correlated with inflammatory changes in the liver.
Other statistically significant changes were not considered test item-related, because these changes were minimal, there were no dose-relationships, and no correlations with microscopic changes and/or observed only in the recovery group.
(see table 3)

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In males, alanine aminotransferase (ALT, 2.80-fold over control), aspartate aminotransferase (AST, 1.46-fold over control), gamma glutamyl transpeptidase (GGT, 8.47-fold over control) and total bilirubin (TBIL, 1.07-fold over control) were increased at 150 mg/kg bw/day. The increases of ALT, GGT and TBIL were statistically significant. In females, ALT (2.05-fold over control), AST (1.43-fold over control), TBIL (1.21-fold over control) and GGT (8.48-fold over control) were also increased at 150 mg/kg bw/day. The increase of GGT was statistically significant. These changes recovered or showed a tendency to recover at the end of the recovery period. Increases of alanine aminotransferase, aspartate aminotransferase, gamma glutamyl transpeptidase and total bilirubin in males and/or females at 150 mg/kg bw/day were considered to be results due to a massive damage of hepatocytes.
Other statistically significant changes were not considered test item-related, because changes were minimal, there were no dose-relationships, no correlated microscopic changes and/or observed only in recovery group.
(see table 4)

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related changes in functional behavior examinations were observed in both sexes during the study.
A statistically significant decrease in grip strength of forelimb in males at 50 and 150 mg/kg bw/day was not considered test item-related since there was no dose-dependency and no change in other parameters during the functional behavioral examinations.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Increased absolute and relative weights of liver (absolute weights: up to 1.27-folds over control, relative weights: up to 1.24-folds over control) and spleen (absolute weights: up to 1.42-folds over control, relative weights: up to 1.37-folds over control) were noted in both sexes at 150 mg/kg bw/day. (see table 4) Also, decreased absolute weights of the left and right testis (88% of control, respectively), and the left and right epididymides (91% of control, respectively) were observed. These changes did not represent a meaningful toxicologic finding as there were no correlated microscopic findings and/or it fully recovered during the recovery period.
Other statistically significant changes were not considered test item-related, because changes were minimal, there were no dose-relationships, no correlated microscopic changes and/or observed only in recovery group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test item-related changes were observed in liver, stomach, spleen and hepatic lymph node of males and/or females at 150 mg/kg bw/day.
In the liver, yellow or dark-red discoloration, yellow focus/foci, enlarged, small, abnormal shape and/or irregular surface of all lobe or specific lobes (caudate, left or median lobe) were observed in both sexes at 150 mg/kg bw/day. After recovery period, a yellow discoloration and/or irregular liver surface were still observed in both sexes at 150 mg/kg bw/day.
In the stomach, adhesion of the stomach (stomach to liver) was observed in one male at the recovery at 150 mg/kg bw/day.
In the spleen, enlargement was observed in one male at 150 mg/kg bw/day. After the recovery period, enlargement was also observed in one female at 150 mg/kg bw/day and white discoloration was observed in one male and female at 150 mg/kg bw/day.
Enlarged hepatic lymph nodes were observed in both sexes at 150 mg/kg bw/day, but they were not observed any more at the end of the recovery period.
Other macroscopic findings noted in the main and the recovery groups were considered to be incidental or spontaneous changes that are commonly seen in SD rats with a given low incidence or severity.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related changes were observed in liver, spleen, hepatic lymph node, and stomach of both sexes at 150 mg/kg bw/day.

Liver:
Slight to marked cholangiofibrosis, minimal to marked hydropic degeneration, minimal to marked necrosis, minimal to marked bile duct hyperplasia, minimal to slight hepatocyte hypertrophy and/or minimal to slight pigmented macrophages in both sexes and slight fibrosis in capsule in one male were observed at 150 mg/kg bw/day. These changes were observed in the periportal region except a fibrosis in capsule. After recovery period, cholangiofibrosis, hydropic degeneration and necrosis were fully recovered and bile duct hyperplasia and hepatocyte hypertrophy showed a tendency of recovery in both sexes at 150 mg/kg bw/day. In the 150 mg/kg bw/day recovery group, minimal to moderate periportal fibrosis in both sexes were also observed and pigmented macrophage was similar to the main groups in both sexes.
Cholangiofibrosis, hydropic degeneration, and necrosis were considered to be an adverse effect of the test-item and are well known and often observed as hepatocellular toxicity caused by various xenobiotics and chemicals. Periportal fibrosis was observed and shown to be a recovery reaction.
Bile duct hyperplasia, hepatocyte hypertrophy, pigmented macrophages and/or fibrosis in the capsule accompanied the above named findings were not considered as an adverse effect at the severity as seen here. These changes were considered to be adaptive changes to a drug-induced increase in a metabolic workload or were considered as secondary findings related to the aforementioned adverse effects in the liver.

Spleen and hepatic lymph node:
Minimal to slight lymphoid hyperplasia in both sexes at 150 mg/kg bw/day and slight chronic inflammation in the capsule in one female at 150 mg/kg bw/day were observed. At the end of the recovery period, lymphoid hyperplasia showed a tendency of recovery in both sexes at 150 mg/kg bw/day and a capsular fibrosis in both sexes and chronic inflammation in capsule were observed in females at 150 mg/kg bw/day. In the hepatic lymph node, moderate lymphoid hyperplasia and slight congestion were observed in both sexes at 150 mg/kg bw/day.
These changes in spleen and hepatic lymph nodes were considered to be associated with a secondary response to the inflammatory changes in the liver.

Stomach:
Minimal to slight squamous cell hyperplasia in the non-glandular region was observed in both sexes at 150 mg/kg bw/day and fully recovered after the recovery period. This change was considered to be non-adverse but an adaptive response following a local irritation caused by the test item since it fully subsided after the recovery period.

Other changes observed in microscopic findings were considered as incidental or spontaneous changes since they were infrequent, generally of low severity, and similarly distributed among the control and the treated groups.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Thyroid hormone anylysis:
No test item-related changes in the thyroid hormone (T4) and the thyroid stimulating hormone (TSH) were observed in the adult male animals.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at 50 mg/kg bw/day

Key result

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

gross pathology

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Table 3. Haematological findings

Group		WBC (x10³/µL)	LYMA (x10³/µL)	MONA (x10³/µL)	LUCA (x10³/µL)	FIB (mg/dL)	PLT (10³/µL)
males
Control	n	12	12	12	12	12	12
	mean	10.45	8.01	0.35	0.12	182.6	994.3
	SD	2.165	1.414	0.134	0.047	9.06	86.03
15 mg/kg bw/day	n	12	12	12	12	12	12
	mean	12.33	9.75	0.44	0.14	186.0	974.8
	SD	2.937	2.488	0.221	0.052	7.16	111.10
50 mg/kg bw/day	n	12	12	12	12	12	12
	mean	11.52	9.07	0.4	0.14	191.8	1118.4*R
	SD	3.175	2.476	0.148	0.075	11.87	82.91
150 mg/kg bw/day	n	12	12	12	12	12	12
	mean	14.64+D	11.76+D	0.50	0.16	223.2+R	1200.6+R
	SD	3.732	3.182	0.265	0.079	30.48	227.41
Recovery group - Control	n	6	6	6	6	6	6
	mean	10.17	7.42	0.39	0.10	199.9	1095.7
	SD	2.447	1.609	0.127	0.041	10.97	88.91
Recovery group 150 mg/kg bw/day	n	6	6	6	6	6	6
	mean	10.93	8.90	0.29	0.12	202.4	1105.0
	SD	1.803	1.311	0.092	0.038	7.12	166.78
females
Control	n	9	9	9	9	9	9
	mean	9.37	6.22	0.47	0.12	216.3	1190.2
	SD	1.673	1.994	0.12	0.049	22.14	236.35
15 mg/kg bw/day	n	12	12	12	12	12	12
	mean	8.66	5.81	0.42	0.11	14.7	1194.0
	SD	2.041	1.726	0.152	0.035	0.46	221.87
50 mg/kg bw/day	n	12	12	12	12	12	12
	mean	9.42	6.08	0.38	0.12	15.2*R	1237.7
	SD	2.808	2.307	0.123	0.050	0.60	139.20
150 mg/kg bw/day	n	11	11	11	11	11	11
	mean	12.14*D	8.55	0.76+D	0.19*R	15.1	1289.4
	SD	2.691	2.644	0.186	0.086	1.03	329.31
Recovery group - Control	n	6	6	6	6	6	6
	mean	6.86	5.42	0.17	0.08	172.1	1093.2
	SD	0.903	0.646	0.071	0.025	6.48	118.97
Recovery group 150 mg/kg bw/day	n	6	6	6	6	6	6
	mean	6.23	4.92	0.20	0.08	181.1*D	1219.7
	SD	1.172	1.042	0.106	0.033	3.84	270.14

R = Dunn Rank Sum Test Significant at the 0.01 level (+) or at 0.05 level (*)

D = Dunnett LSD Test Significant at the 0.01 level (+) or at 0.05 level (*)

WBC = total leukocyte count

LYMA = absolute lymphocyte count

MONA = absolute monocyte count

LUCA = absolute large unstained cells counts

FIB = fibrinogen

PLT = platelet count

Table 3. Clinical biochemistry findings

Group		ALT (IU/L)	AST (IU/L)	GGT (IU/L)	TBIL (mg/dL)
males
Control	n	12	12	12	12
	mean	44.3	172.6	0.49	0.183
	SD	42.60	83.42	0.497	0.1209
15 mg/kg bw/day	n	12	12	12	12
	mean	34.4	149.4	0.41	0.152
	SD	11.40	35.09	0.293	0.0270
50 mg/kg bw/day	n	12	12	12	12
	mean	34.9	152.0	0.73	0.150
	SD	6.52	16.69	0.308	0.0234
150 mg/kg bw/day	n	12	12	12	12
	mean	124.0*R	251.3	4.15+R	0.195*R
	SD	181.65	216.19	6.111	0.0341
Recovery group - Control	n	6	6	6	6
	mean	47.4	168.5	0.44	0.131
	SD	26.76	40.47	0.148	0.0105
Recovery group 150 mg/kg bw/day	n	6	6	6	6
	mean	53.8	162.5	0.80+D	0.140
	SD	12.46	36.32	0.207	0.0290
females
Control	n	9	9	9	9
	mean	65.5	181.8	1.18	0.118
	SD	22.29	46.30	0.747	0.0227
15 mg/kg bw/day	n	12	12	12	12
	mean	59.8	164.5	0.78	0.122
	SD	15.82	32.37	0.355	0.0178
50 mg/kg bw/day	n	12	12	12	12
	mean	70.1	162.1	1.45	0.135
	SD	16.19	27.38	0.564	0.0237
150 mg/kg bw/day	n	11	11	11	11
	mean	134.0	259.5	10.01+R	0.143
	SD	176.91	210.57	8.246	0.0336
Recovery group - Control	n	6	6	6	6
	mean	44.1	142.8	1.08	0.197
	SD	10.60	25.76	0.252	0.0289
Recovery group 150 mg/kg bw/day	n	6	6	6	6
	mean	43.2	190.2+D	1.15	0.175
	SD	13.61	25.34	0.425	0.0341

R = Dunn Rank Sum Test Significant at the 0.01 level (+) or at 0.05 level (*)

D = Dunnett LSD Test Significant at the 0.01 level (+) or at 0.05 level (*)

ALT = alanine aminotransferase

AST = aspartate aminotransferase

GGT = gamma glutamyl transpeptidase

TBIL = total bilirubin

Table 4. Findings in organ weight

Group		Liver absolut (g)	Liver relative (g/kg bw)
males
Control	n	12	12
	mean	13.623	2.7296
	SD	1.8269	0.17755
15 mg/kg bw/day	n	12	12
	mean	13.787	2.7626
	SD	1.2970	0.21793
50 mg/kg bw/day	n	12	12
	mean	14.437	2.8353
	SD	1.7329	0.19210
150 mg/kg bw/day	n	12	12
	mean	16.385+D	3.3854+D
	SD	1.5829	0.23912
Recovery group - Control	n	6	6
	mean	14.781	2.7965
	SD	1.1645	0.19102
Recovery group 150 mg/kg bw/day	n	6	6
	mean	15.223	2.8543
	SD	1.2307	0.20696
females
Control	n	9	9
	mean	12.366	3.7479
	SD	1.3052	0.33675
15 mg/kg bw/day	n	12	12
	mean	12.924	3.8280
	SD	0.8599	0.21893
50 mg/kg bw/day	n	12	12
	mean	12.632	3.7100
	SD	1.0564	0.18965
150 mg/kg bw/day	n	11	11
	mean	15.757+R	4.5949+R
	SD	2.2078	0.52216
Recovery group - Control	n	6	6
	mean	8.684	2.8012
	SD	0.5044	0.06238
Recovery group 150 mg/kg bw/day	n	6	6
	mean	9.687*D	3.1183+R
	SD	0.7912	0.18695

R = Dunn Rank Sum Test Significant at the 0.01 level (+) or at 0.05 level (*)

D = Dunnett LSD Test Significant at the 0.01 level (+) or at 0.05 level (*)    

                 

Conclusions:

The NOAEL for systemic toxicity was considered to be 50 mg/kg bw/day in both sexes, based on cholangiofibrosis, hydropic degeneration and necrosis of liver in both sexes at 150 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable study (Klimisch score 1), and is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.6, of Regulation (EC) No 1907/2006.

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

The test substance was tested in a combined repeated dose oral toxicity study with the reproduction/developmental toxicity screening study according to OECD Guideline 422 and in compliance with GLP (2018). Twelve Sprague Dawley rats per sex and dose were treated via gavage with the test substance at doses of 15, 50 and 150 mg/kg bw/day, respectively. The control group received the vehicle corn oil. Additionally, non-mated recovery groups of 6 rats per sex were allocated to the control and high dose groups. Males and females of main groups were dosed for two weeks prior to mating and continued through the day before sacrifice in males (at least 50 days), and continued through the lactation day (LD) 13 in females. The high-dose recovery group was assigned to a two week treatment free period. The dose levels were selected based on the results of a repeated dose 2-week dose range-finding study in which doses of 15, 60 and 240/150 mg/kg bw/day had been tested. In that study, one found dead female was observed on treatment day 5 at a dose level of 240 mg/kg bw/day. The dose level of the high dose group was changed from 240 to 150 mg/kg bw/day on treatment day 6. Increased alanine aminotransferase (AST), alanine aminotransferase (ALT) and yellow discoloration of liver were observed in males at 60 mg/kg bw/day. At 240/150 mg/kg bw/day, general toxicity including soft feces, lacrimation and soiled fur in males, and decreased body weight gain and food consumption in both sexes were observed. In addition, increased AST and ALT levels were observed in males. Absolute and relative spleen weights were increased in both sexes.

In the main study, general systemic observations including mortality, general clinical signs, body weights, body weight gain, food consumption, functional behavior examination, motor activity examination, macroscopic findings, hematology, coagulation, clinical chemistry, organ weights and microscopic findings were conducted. Thyroid hormone (T4) level in blood was also analyzed for adult males at sacrifice.

No test substance-related deaths or moribundity occurred in both sexes of all groups throughout the study.  

In general systemic observations, test item-related salivation was observed in both sexes at all test item treatment groups, however, and it was not considered to have any toxicological significance since it was considered to be attributed to the palatability of the test item.

In hematology, total leukocyte count (WBC, up to 1.40-fold over VC), absolute lymphocyte count (LYMA, up to 1.47-fold over VC), monocyte count (MONA, up to 1.62-fold over VC) and large unstained cells counts (LUCA, up to 1.58-fold over VC) were increased in both sexes at 150 mg/kg bw/day. Fibrinogen (FIB, 1.22-fold over VC) in males at 150 mg/kg bw/day and platelet count (PLT, up to 1.21-folds over VC) in males at 50 and 150 mg/kg bw/day were also increased. In clinical chemistry, alanine aminotransferase (ALT, up to 2.80-fold over VC), aspartate aminotransferase (AST, up to 1.46-fold over VC), gamma glutamyl transpeptidase (GGT, up to 8.48-fold over VC) and total bilirubin (TBIL, up to 1.21-fold over VC) were increased in both sexes at 150 mg/kg bw/day.

Absolute and relative weights of liver (up to 1.27-folds and 1.24-folds over VC, respectively) and spleen (up to 1.42-folds and 1.37-folds over VC, respectively) in both sexes at 150 mg/kg bw/day were increased and absolute weights of testes (88% of VC) and epididymides (91% of VC) in males at 150 mg/kg bw/day were decreased. These changes were recovered or showed tendency of recovery after 2 weeks of recovery period. 

In the macroscopic findings, yellow or dark-red discoloration, yellow focus/foci, enlarged, small, abnormal shape and/or irregular surface in liver and enlarged spleen and hepatic lymph node were observed in males or females at 150 mg/kg bw/day. After the recovery period, yellow discoloration and irregular surface of the liver were observed in both sexes at 150 mg/kg bw/day. The spleen was enlarged in females and showed white discoloration in both sexes at 150 mg/kg bw/day. Enlarged hepatic lymph nodes were not observed in the recovery groups.

In the microscopic findings, test item-related changes were observed in liver, spleen, hepatic lymph nodes and stomach of both sexes at 150 mg/kg bw/day. Cholangiofibrosis, hydropic degeneration, necrosis, bile duct hyperplasia, hepatocyte hypertrophy, and/or pigmented macrophages in both sexes and fibrosis in capsule in males were observed in the liver. Cholangiofibrosis, hydropic degeneration and necrosis were considered to be test item-related adverse effects. After the recovery period, cholangiofibrosis, hydropic degeneration and necrosis were fully recovered and bile duct hyperplasia and hepatocyte hypertrophy showed a tendency of recovery in both sexes. In the recovery groups, fibrosis in periportal region and pigmented macrophage were observed in both sexes. Lymphoid hyperplasia in spleen and hepatic lymph nodes in both sexes at 150 mg/kg bw/day bw/day, chronic inflammation in the spleen of one female at 150 mg/kg bw/day were observed associated with microscopic changes in the liver. After the recovery period, lymphoid hyperplasia of the spleen showed a tendency of recovery in females. Also, capsular fibrosis in both sexes and chronic inflammation in females were observed in the spleen after the recovery period. In the stomach, squamous cell hyperplasia in the non-glandular region in both sexes at 150 mg/kg bw/day was observed and considered to be a local adaptive response to the oral administration of the test-item. Squamous hyperplasia was fully recovered after recovery period.

In conclusion, cholangiofibrosis, hydropic degeneration and necrosis of the liver observed in both sexes at 150 mg/kg bw/day were considered to be adverse and test-substance related. Therefore, the NOAEL for general toxicity was considered to be 50 mg/kg/day for both sexes.

 

Justification for classification or non-classification

The LOAEL in the 422 study was 150 mg/kg bw/day. In this study toxic effects on the liver were observed, which are most probably caused by allyl alcohol which can be generated by ester hydrolysis catalysed by unspecific esterases. The liver effects were largely reversible during the 2 -week recovery period. Thus, at the dose level of 150 mg/kg bw/day no severe functional impairment of liver function was observed. In conclusion, the severity level requring a STOR-RE category 2 classification was not reached. While the available study was not a 90 -day study, its duration was considerably longer than 28 days. Moreover, experiments with another allyl ester, 2 -propenyl-heptanoic, CAS 142 -19 -8, revealed similar toxic effects on the liver which were especially prominent after bolus dose administration, i.e. the NOAEL of a 90 -day feeding study was not lower then the NOAEL from a 28 -day gavage study. Since human exposure to allyl phenoxy acetate is not associated with bolus exposure, It is not considered necessary to include a time extrapolation factor in the evaluation of the STOT-RE classification. In conclusion, the available data on repeated dose toxicity of the test substance do not meet the criteria for classification as STOT-RE 2 (H373) according to Regulation (EC) No 1272/2008.