2-(2,6-diethyl-4-methyl-phenyl)propanediamide

Endpoint summary

Description of key information

Oral: NOAEL = 150 mg/kg/day, 90 days, male/female rat, OECD 408, King 2016

Oral: NOAEL = 150 mg/kg/day, 28 days, male/female rat, OECD 407, Milburn 2004

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Remarks:

The oral sub-chronic toxicity study was conducted solely to comply with a non-EU national registration requirement, and has been provided here in accordance with REACH, Article 22(1)e.

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 October 2015 - 28 April 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on species / strain selection:

Commonly used rodent species

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, CT9 4LT, England.
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: 7-8 weeks
- Weight at study initiation: 201-272 g (males), 136-180 g (females)
- Fasting period before study: No
- Housing: Groups of five, by sex, in grid-floor cages suspended over paperlined trays.
- Diet: Pelleted diet, LabDiet 5L0S EURodent Diet (manufactured by PMI Nutrition International), supplied by International Product Supplies, ad libitum
- Water: Mains tap water ad libitum
- Acclimation period: 13 days

DETAILS OF FOOD AND WATER QUALITY: Certificates of analysis for diet and water were retained within the Sequani archives. It was considered that none of the contaminants that were monitored was present at a level that might have prevented the study objective from being achieved.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 40-70
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 21 October 2015 To: 28 April 2016

Route of administration:

oral: gavage

Details on route of administration:

Animals were dosed once daily for at least 90 days, by gavage using a rubber catheter and disposable syringe at a constant dose volume of 10 mL/kg body weight until necropsy. Individual doses were adjusted according to the most recent body weight.

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5 % (w/v) aqueous carboxymethylcellulose with 0.1 % (v/v) Tween 80

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS: The test item was formulated at intervals within known stability for each group separately, as a suspension in the vehicle. The required amount of test item was weighed into a container and a small quantity of vehicle was initially added to create a smooth paste using a spatula. After further addition of vehicle and mixing, the resultant suspension was made up to final volume or weight with vehicle, rinsing the spatula in the process and mixed. Formulations were then placed in an ultrasonic bath for a minimum of 15 minutes (in short bursts, as necessary, to prevent heating of the formulation) to break up any remaining large particulate. Formulations were divided into daily aliquots and were stored refrigerated (2-8°C), before use. Test item formulations were stirred for at least 15 minutes before the start of dosing until the completion of their use for dosing, to ensure thorough re-suspension and homogeneity.

- VEHICLE
- Justification for use and choice of vehicle (if other than water): Not reported

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Formulations of the test substance in 0.5 % w/v aqueous carboxymethylcellulose with 0.1 % v/v Tween 80 at concentrations between 1 and 150 mg/mL, spanning those used in this study (1.5 mg/mL to 100 mg/mL), have been shown to be stable for up to six days when stored at room temperature, up to 12 days when stored refrigerated and one month when stored frozen.
Samples were taken from each formulation. Formulations prepared for use on Day 1 were analysed to assess their homogeneity and achieved concentrations. Having satisfactorily confirmed homogeneity on Day 1, subsequent formulations for use during Weeks 4, 8 and 12 were analysed to determine achieved concentrations only. Samples from vehicle used to dose Controls on Day 1 and during Weeks 4, 8 and 12 were analysed to confirm absence of test item. All samples were analysed for test substance using a validated method.
Results: Mean achieved concentrations of the test item formulations used to dose animals on Day 1 and during Weeks 4, 8 and 12 of the study were within 3% of nominal with coefficients of variation no greater than 2.5%, meeting the acceptance criteria (± 10% and ≤ 5%, respectively). No test substance was detected in the vehicle used to dose Control animals. It is considered, therefore, that formulations were both homogeneous and accurately prepared.

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

150 mg/kg bw/day (nominal)

Dose / conc.:

15 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected in consultation with the Sponsor on the basis of results from a previous 28 day study in the rat. In this study, groups of five male and five female rats were dosed with 0, 15, 150, or 1000 mg/kg/day test substance. There were no test item effects on mortality, body weight, body weight gain, food intake or FOB measurements at any dose level. The liver was identified as the target organ and there was a dose-related increase in liver weight at 150 and 1000 mg/kg/day in males and females and changes in blood chemistry parameters in the females. A similar outcome was expected after 90 days dosing: the low dose level of 15 mg/kg/day was intended to be the NOEL and the high dose level of 1000 mg/kg/day was selected as the maximum permissible dose level for studies of this design.
- Rationale for animal assignment : Animals were allocated to groups using a stratified body weight randomisation procedure based on individual body weights recorded on arrival.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes (Mortality and morbidity)
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily and, in addition, for the first four weeks of treatment, animals were observed before, shortly after and about one hour after dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: Daily for the first week and weekly thereafter

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Daily for the first week and weekly thereafter

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pre-treatment (all rats), week 13 (control and high dose rats)

FUNCTIONAL OBSERVATIONS: Yes
- Time schedule for examinations: All animals were observed once weekly, starting pre-dose, for their behaviour both within their cage and then after placement in an open arena. On one occasion during Week 13 of the study, sensorimotor responses to visual, acoustic, tactile or proprioceptive stimuli, grip strength and motor activity were recorded for all animals.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 13
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Not specified
- How many animals: All
- The following parameters were examined.:haemoglobin concentration (Hb), total leucocyte count (WBC), red blood cell count (RBC), neutrophils (Neut), packed cell volume (PCV), lymphocytes (Lymph), mean cell volume (MCV), monocytes (Mono), mean cell haemoglobin (MCH), eosinophils (Eosin), mean cell haemoglobin concentration (MCHC), basophils (Baso), red blood cell distribution width (RDW), large unstained cells (LUC), platelet count (Plate), reticulocytes (Retics), absolute reticulocyte count (A retics), prothrombin time (PT), activated partial thromboplastin time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 13
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Not specified
- How many animals: All
- The following parameters were examined: urea, globulin (Glob), creatinine (Cren), albumin/globulin ratio (A/G), glucose (Gluc), total bilirubin (BiliT), alkaline phosphatase (ALP), cholesterol (Chol), alanine aminotransferase (ALT), triglyceride (Trigs), aspartate aminotransferase (AST), sodium (Na), gamma glutamyl transpeptidase (GGT), chloride (Cl), bile acids (B.Acids), calcium (Ca), total protein (T.Prot), potassium (K), albumin (Alb), inorganic phosphorus (I. Phos).

URINALYSIS: No

IMMUNOLOGY:No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- At the end of the treatment period all animals were killed by exposure to carbon dioxide gas in a rising concentration. All animals were weighed and examined externally. The abdominal cavity was opened and the animals were exsanguinated from the caudal vena cava. The cranial and thoracic cavities were opened and a full internal examination was performed.
ORGAN WEIGHTS: The following organs were weighed after trimming of fat and other contiguous tissue (contralateral organs were weighed together): adrenal glands, prostate & seminal vesicles (incl. coagulating gland), brain, spleen, epididymides, testes, heart, thymus, kidneys, thyroids (including parathyroids), liver, uterus (including cervix and oviducts), ovaries.

HISTOPATHOLOGY: Yes
- Initially, all the following tissues from Control and high dose animals (in addition to gross lesions from all groups) were examined. Subsequently (due to microscopic findings in the liver, thyroids, adrenals and ovaries of high dose animals), the liver and thyroids from both sexes, adrenals from males and ovaries from females were also examined from the low and intermediate dose groups.
- tissues examined: adrenal glands, pancreas, aorta, pharynx (laryngeal), bone marrow smear, pituitary, brain (3 levels examined), prostate & seminal vesicles (incl. coagulating gland), caecum, colon, rectum, duodenum, salivary gland (submandibular), epididymides, sciatic nerve, eyes (including optic nerves), skeletal muscle, femur & joint (including marrow), skin (with mammary gland), Harderian glands, spinal cord (3 levels examined LS and TS), heart, spleen, ileum, sternum with bone marrow, Jejunum (including Peyer’s patch), stomach, kidneys (one LS and one TS), submandibular lymph nodes, lacrimal glands, testes, larynx, thymus, liver, thyroids (including parathyroids), lungs (including mainstem bronchi), tongue, mammary gland (with inguinal skin), trachea, mesenteric lymph nodes, urinary bladder, nasal cavity (one section - 3rd level), uterus (including uterine cervix and oviducts), oesophagus, vagina, ovaries, all gross lesions.

Statistics:

Data were processed to give group mean values and standard deviations, where appropriate. Where the data allowed, the following methods were used for statistical analysis, comparing Groups 2, 3 and 4 against Group 1. All statistical tests were two-sided with minimum significance levels of 5 % and 1 %. Non-parametric statistics were not routinely conducted. When used, Dunnett’s test was conducted regardless of the outcome of the analysis of variance (ANOVA) or analysis of covariance (ANCOVA). Body weight, cumulative body weight gain from the start of dosing, food consumption, haematology, coagulation and blood chemistry parameters, grip strength and motor activity data were analysed using a parametric ANOVA. Organ weights were analysed using ANOVA for the absolute weights and by analysis of covariance (ANCOVA) using terminal kill body weight as covariate. For all of the parameters evaluated initially by ANOVA or ANCOVA, Dunnett's test was used to compare the Control and treated groups, based on the error mean square in the ANOVA or ANCOVA. The Dunnett's test was performed for all continuous data parameters, regardless of whether the initial ANOVA or ANCOVA was statistically significant. Qualitative functional observational battery parameters or any other parameters not mentioned above were not analysed statistically.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation throughout the treatment period for both sexes given 1000, 150 or 15 mg/kg/day. The number and frequency of these occasions increased in a dose related manner.
Yellow stained bedding was observed in cages for both sexes given 1000 mg/kg/day, and for males given 150 mg/kg/day.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Throughout the study females given 1000 mg/kg/day consistently gained statistically significantly more weight than Controls (p<0.01 to p<0.05), such that their cumulative mean body weight gains and mean body weights at the end of the dosing period were 30% and 10% higher, respectively, than the Control values. There were no effects on body weight in males at 1000 mg/kg/day or in either sex at 15 or 150 mg/kg/day.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A marginal increase in mean food intake was observed for one out of two cages of females given 1000 mg/kg/day during the study. There was no effect on food intake during the study for males given any dose level and females given 15 or 150 mg/kg/day.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day females had a statistically significant increase in total white blood cell count due to an increase in neutrophils and monocytes (p<0.01 to p<0.05). Males given 1000 or 150 mg/kg had a statistically significant increase in platelet count and reduced prothrombin time (p<0.01), compared with Controls. There were no other effects on haematology or coagulation parameters.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

There was a marked, test item-related, approximately four fold increase (p<0.01) in mean plasma bile acid concentration in females given 1000 mg/kg/day and a slight non-significant increase in males from this group, compared with Controls. Aspartate aminotransferase activity was lower for both sexes given 1000 mg/kg/day (p<0.01) and for females given 15 or 150 mg/kg/day (p<0.05). Males given 1000 mg/kg/day also had low alkaline phosphatase activity (p<0.01) when compared with Controls. Group mean total plasma protein was increased (with high levels of albumin and globulin for males and only globulin for females) and associated albumin/globulin ratio was low for both sexes given 1000 mg/kg/day compared with Controls. Cholesterol was increased in both sexes given 1000 mg/kg/day (p<0.01 to p<0.05), compared with Controls. Coupled with the microscopic findings in the liver of high dose animals, these changes in this group were considered to be test item-related. Females given 1000 mg/kg/day also showed increase in inorganic phosphorus and triglyceride concentration (p<0.01 to p<0.05). Plasma chloride was slightly lower for both sexes given 1000 mg/kg/day (p<0.01 to p<0.05). Where statistically significant differences were observed for other blood chemistry parameters these differences were minimal and were considered to reflect normal biological variation.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

All treated females were more active and travelled a greater distance than Controls for the first and second 10 minutes of motor activity monitoring, achieving statistical significance (p<0.01 to p<0.05) only in animals given 1000 mg/kg/day. There were no differences from Controls for subsequent intervals. There was no effect in the males at any dose level. Forelimb grip strength was significantly (p<0.05) reduced for males given 1000 mg/kg/day, although their hind limb grip strength was similar to Controls. There were no effects on grip strength for females given 1000 mg/kg/day or either sex at any other dose level. There were no test item-related effects on functional observations or on sensorimotor responses to visual, acoustic, tactile or proprioceptive stimuli at any dose level.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Group mean absolute and adjusted liver weights were higher than Controls for both sexes given 1000 mg/kg/day (39% and 41% for males, and 45% and 37% for females, respectively) and for males given 150 mg/kg/day (11%) (p<0.01). However, all relative liver weights for males given 150 mg/kg/day were well within the laboratory background ranges. Group mean adjusted thyroids weights were statistically significantly higher than Controls for all treated males (21%, 24% and 35% in the groups given 15, 150 or 1000 mg/kg/day respectively (p<0.01 to p<0.05)); mean absolute thyroid weight was also higher than the Control mean in these animals. Group mean absolute and adjusted thyroids weights were also higher than Controls (13% and 4% respectively) for females given 1000 mg/kg/day, but the differences did not achieve statistical significance. However, most relative thyroid weights for females in this group were at the upper limit of the laboratory background ranges and that for one female exceeded the normal upper limit. Group mean absolute and adjusted adrenal weights were statistically significantly lower (-16%; p<0.01) than Controls in female rats given 150 mg/kg/day. For females given 1000 mg/kg/day the group mean adjusted adrenal weights were higher than Control mean (4%). Other statistically significant inter-group differences were considered to reflect normal biological variation rather than any effect of the test substance.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Enlarged adrenals were noted in five out of ten females given 1000 mg/kg/day compared with one affected Control female. There was no microscopic correlation of this macroscopic finding and therefore the macroscopic findings were considered to reflect the inter-animal variation of normal aging changes in the female rat. A variety of spontaneous macroscopic changes was noted in Control and treated animals with no indication of an effect of treatment. The spectrum of these findings is generally consistent with changes encountered in rats of this age kept under laboratory conditions.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Liver: Test item-related centrilobular hepatocyte hypertrophy (minimal or slight) was found in males and females given 1000 mg/kg/day. There was no such change in animals given 15 or 150 mg/kg/day.
Thyroids: Follicular cell hypertrophy (minimal) was found in males and females given 1000 mg/kg/day but not in animals given 15 or 150 mg/kg/day.
Adrenals: Vacuolation of the zona fasciculata and zona glomerulosa was found only in males given 1000 mg/kg/day but not in males given 15 or 150 mg/kg/day.
Ovaries: Hypertrophy and vacuolation of the interstitial gland was found in most of the females given 1000 mg/kg/day but not in females given 15 or 150 mg/kg/day.
A variety of spontaneous microscopic changes was noted in Control and treated animals with no indication of an effect of treatment. The spectrum of these findings is generally consistent with changes encountered in rats of this age kept under laboratory conditions.

Key result

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: Ovarian changes in females and adrenal changes in males at 1000 mg/kg/day. Significant increases in liver and thyroid weights in the presence of histopathological correlates were observed in males and females at this dose level.

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

female reproductive system

Organ:

ovary

Treatment related:

yes

Dose response relationship:

no

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

endocrine system

Organ:

adrenal glands

Treatment related:

yes

Dose response relationship:

no

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

endocrine system

Organ:

thyroid gland

Treatment related:

yes

Dose response relationship:

no

Table 1: Intergroup comparison of body weight gain (g)-selected timepoints

	Dose level (mg/kg/day)
	Males				Females
Day	0	15	150	1000	0	15	150	1000
1-2	3.9	4.9	5.4	6.0	3.8	2.8	3.4	1.4
1-4	16.6	17.2	18.9	18.8	10.7	7.7	8.4	11.0
1-8	36.0	37.5	39.3	39.1	20.1	18.1	20.8	25.7
1-22	90.2	88.3	95.8	91.4	47.0	46.8	47.3	61.0**
1-50	167.4	156.8	167.9	167.9	80.3	80.3	79.2	97.9**
1-71	198.4	182.7	195.6	196.7	88.7	91.7	89.6	108.1**
1-92	223.8	206.7	216.7	219.2	92.8	98.5	97.7	120.7**

* Statistically significant difference from control group mean, p<0.05

** Statistically significant difference from control group mean, p<0.01

 

Table 2: Intergroup comparison of food consumption (g/animal/day) -selected timepoints

	Dose level (mg/kg/day)
	Males				Females
Day	0	15	150	1000	0	15	150	1000
1-2	22.4	21.9	23.8	19.5	15.9	14.7	15.3	11.2
3-4	22.4	22.3	24.1	22.5	17.4	14.9	15.7	16.3
7-8	24.7	24.4	26.2	23.2	17.4	18.3	18.6	17.8
15-22	24.4	24.3	26.6	24.7	20.4	18.7	19.3	20.6
43-50	24.7	24.0	26.3	23.9	17.9	19.1	19.0	20.5
64-71	25.2	25.6	24.4	24.9	19.4	19.3	18.7	20.1
85-92	24.7	24.5	25.9	23.8	17.1	18.4	18.4	19.3
1-92	25.0	24.7	26.3	24.7	18.2	19.0	18.8	19.9

No statistically significant differences from control group mean

 

Table 3: Intergroup comparison of selected haematology parameters

Parameter	Dose level (mg/kg/day)
	Males				Females
	0	15	150	1000	0	15	150	1000
WBC (10^3/µL)	6.965	6.043	6.391	7.215	4.499	4.752	5.232	6.115*
Neut (%)	12.43	13.03	11.19	13.82	12.67	12.30	11.32	12.41
Mono (%)	1.57	1.66	1.39	1.73	1.98	2.10	2.25	2.94*
Plate (10^3/µL)	836.7	844.0	910.2	996.8**	840.6	867.5	817.5	891.1
PT (sec)	23.38	23.50	21.69**	20.61**	21.81	22.23	22.21	20.93

* Statistically significant difference from control group mean, p<0.05

** Statistically significant difference from control group mean, p<0.01

 

Table 4: Intergroup comparison of selected clinical chemistry parameters

Parameter	Dose level (mg/kg/day)
	Males				Females
	0	15	150	1000	0	15	150	1000
B. Acids (µmol/L)	9.33	7.34	9.58	10.77	11.41	7.44	12.44	45.64**
AST (U/L)	56.8	56.2	55.1	49.1**	85.3	56.4*	52.8*	50.8**
T. prot (g/dL)	6.40	6.50	6.65*	7.21**	7.11	7.16	7.01	7.20
Alb (g/dL)	4.03	4.10	4.18	4.19**	4.94	4.93	4.74	4.56**
Glob (g/dL)	2.37	2.40	2.47	3.02**	2.17	2.23	2.27	2.64**
A/G ratio	1.71	1.71	1.70	1.39**	2.28	2.22	2.09**	1.73**
Chol (mg/dL)	55.5	53.1	62.3	69.6*	48.7	59.4*	58.4	81.8**
I. Phos (mg/dL)	5.00	4.89	5.11	4.70	3.90	4.11	4.47	4.90**
Trigs (mg/dL)	132.1	117.8	131.1	132.9	94.7	97.1	89.9	153.5*
Cl (nmol/L)	100.4	100.8	100.2	98.7*	100.6	100.2	99.7	97.1**

* Statistically significant difference from control group mean, p<0.05

** Statistically significant difference from control group mean, p<0.01

 

Table 5: Intergroup comparison of motor activity

Parameter	Dose level (mg/kg/day)
	Males				Females
	0	15	150	1000	0	15	150	1000
Movement (a)
1	899.2	1004.7	993.6	1272.5	1069.4	1509.3	1454.6	1642.8**
2	638.6	544.9	589.9	689.5	633.4	756.1	842.2*	880.1*
3	513.0	299.8	510.7	486.6	448.7	482.1	673.6	403.5
4	532.2	402.8	593.3	534.5	414.9	373.1	415.8	256.0
5	345.0	228.0	397.5	418.1	368.9	353.4	217.5	207.1
6	280.8	116.9	298.7	230.6	152.3	314.7	256.8	282.8
Distance (b)
1	3405.8	3758.2	3762.6	4679.8	3886.6	5300.8*	5154.1	5873.6**
2	2400.0	2006.3	2157.0	2527.4	2352.1	2688.2	2977.1	3078.8*
3	1948.7	1082.4	1858.3	1769.5	1642.4	1730.9	2395.6	1414.5
4	1976.3	1532.4	2215.3	1936.2	1579.7	1356.0	1534.6	931.8
5	1289.2	872.2	1483.0	1530.3	1318.9	1294.8	745.5	700.7
6	1032.6	466.4	1034.4	866.2	581.9	1130.5	954.7	1037.5
At rest (c)
1	291.4	274.2	285.5	228.0	272.1	223.4	234.1	195.0**
2	358.5	391.9	391.7	359.1	364.5	357.1	356.8	330.9
3	398.1	473.6	422.4	420.6	427.5	436.5	395.2	470.4
4	406.9	445.2	387.6	407.1	435.3	469.8	447.4	510.5
5	456.4	496.9	452.6	455.5	460.7	460.8	520.8	531.7
6	482.1	540.1	496.1	513.4	529.3	483.2	501.6	502.9

(a) movement counts over 10 min periods (b) distance travelled (cm) over 10 min periods (c) time at rest (sec) over 10 min periods

* Statistically significant difference from control group mean, p<0.05

** Statistically significant difference from control group mean, p<0.01

 

Table 6: Intergroup comparison of forelimb grip strength

Dose level (mg/kg/day)
Males				Females
0	15	150	1000	0	15	150	1000
511.87	470.73	461.13	402.30*	419.53	360.70	394.40	390.07

* Statistically significant difference from control group mean, p<0.05

 

Table 7: Intergroup comparison of selected organ weights (g)

		Dose level (mg/kg/day)
		Males				Females
Organ		0	15	150	1000	0	15	150	1000
Liver	actual	14.001	13.949	15.586	19.445**	8.265	8.654	8.885	12.008**
	adjusted	13.793	14.329	15.375**	19.484**	8.485	8.644	8.992	11.656**
Thyroid	actual	0.0192	0.0229	0.0238*	0.0257**	0.0191	0.0180	0.0175	0.0215
	adjusted	0.0191	0.0232*	0.0237*	0.0257**	0.0197	0.0181	0.0178	0.0205
Adrenal	actual	0.0683	0.0643	0.0694	0.0691	0.0883	0.0814	0.0745*	0.0941
	adjusted	0.0676	0.0655	0.0687	0.0692	0.0890	0.0815	0.0749*	0.0929

* Statistically significant difference from control group mean, p<0.05

** Statistically significant difference from control group mean, p<0.01

 

Table 8: Intergroup comparison of treatment-related histopathological findings in liver

Finding		Dose level
		M	M	M	M	F	F	F	F
		0	15	150	1000	0	15	150	1000
No. tissues examined		10	10	10	10	10	10	10	10
Hyper- trophy, hepato- cyte, centril- obular	min.	0	0	0	2	0	0	0	0
	slight	0	0	0	8	0	0	0	10
	total	0	0	0	10	0	0	0	10

Table 9: Intergroup comparison of treatment-related histopathological findings in thyroids

Finding		Dose level
		M	M	M	M	F	F	F	F
		0	15	150	1000	0	15	150	1000
No. tissues examined		10	10	10	10	10	10	10	10
Hyper- trophy, follicular cell	min.	0	0	0	9	0	0	0	7
	total	0	0	0	9	0	0	0	7

 

Table 10: Intergroup comparison of treatment-related histopathological findings in adrenals

Finding		Dose level
		M	M	M	M
		0	15	150	1000
No. tissues examined		10	10	10	10
Vacuolation, fasciculate/glomerulosa	min.	0	0	0	4
	slight	0	0	0	4
	total	0	0	0	8

Table 11: Intergroup comparison of treatment-related histopathological findings in ovaries

Finding		Dose level
		0	15	150	1000
No. tissues examined		10	10	10	10
Hypertrophy/vacuolation, interstitial gland	min.	0	0	0	2
	slight	0	0	0	2
	mod.	0	0	0	3
	total	0	0	0	7

 

Conclusions:

The test substance administered once daily by oral gavage to the rat at doses of 0, 15, 150 or 1000 mg/kg/day was tolerated for at least 90 days, but elicited test-item related ovarian changes in females and adrenal changes in males at 1000 mg/kg/day. In the absence of any functional investigations the adverse nature of these changes remains equivocal. In addition, significant increases in liver and thyroid weights in the presence of histopathological correlates were observed in males and females at this dose level. The No Observed Adverse Effect Level (NOAEL) for both sexes was considered to be 150 mg/kg/day.

Executive summary:

The oral repeated dose toxicity of the substance to the rat was investigated in a GLP study according to the OECD TG 408. Groups of 10 male and 10 female Crl:WI(Han) rats were dosed with 0 (vehicle), 15, 150 or 1000 mg/kg/day test substance, once daily by gavage, for at least 90 days, until the day before necropsy. All animals were observed daily from the start of treatment. Body weights and food intake were recorded daily for the first week of treatment and weekly thereafter. The eyes of all animals were examined before the start of treatment, with Control and high dose animals also examined in Week 13. Blood samples were taken during Week 13 for clinical pathology assessment. All animals were subjected to standard arena observations pre-dose and once weekly thereafter; in addition, grip strength, motor activity and sensorimotor responses to visual, acoustic, or proprioceptive stimuli were assessed once in Week 13. All animals were subjected to a gross necropsy, specified organs were weighed and a full list of tissues was examined microscopically from the Control and high dose animals. In addition, the liver and thyroids from both sexes, adrenals from males and ovaries from females were examined microscopically from intermediate and low dose animals.

There were no deaths during the study. Excessive salivation was seen after dosing on a number of occasions throughout the treatment period for both sexes given 15, 150 or 1000 mg/kg/day, number and frequency of these occasions increased in a dose related manner. Yellow stained bedding was observed in cages for males given 150 mg/kg/day and both sexes given 1000 mg/kg/day. Throughout the study females given 1000 mg/kg/day consistently gained more weight than Controls. Their cumulative mean body weight gains and mean body weights at the end of the dosing period were 30% and 10% higher, respectively, than the Control values. There were no effects on body weight in males at 1000 mg/kg/day or in either sex at 15 or 150 mg/kg/day. A slight increase (+9%) in mean food intake was observed for females given 1000 mg/kg/day during the study. There was no notable effect on food intake during the study for males given any dose level and females given 15 or 150 mg/kg/day. There were no test item-related effects on functional observations or on sensorimotor responses to visual, acoustic, tactile or proprioceptive stimuli at any dose level. All treated females were more active and travelled a greater distance than Controls for the first and second 10 minutes of motor activity monitoring, which was more notable for animals given 1000 mg/kg/day but with no differences from Controls for subsequent intervals. Forelimb grip strength was reduced for males given 1000 mg/kg/day, but their hind limb grip strength was similar to Controls. There were no effects on grip strength for females given 1000 mg/kg/day or either sex at any other dose level. There were no ocular changes that were considered to be related to test substance administration. At 1000 mg/kg/day females had a statistically significant increase in white blood cells count due to increase in neutrophils and monocytes. Males had statistically significant increase in platelets and reduced prothrombin time, compared with Controls. Bile acids were notably increased for females given 1000 mg/kg/day. Activity of aspartate aminotransferase was lower for both sexes given 1000 mg/kg/day and for females given 15 or 150 mg/kg/day. Males given 1000 mg/kg/day also had low alkaline phosphatase activity when compared with Controls. Group mean total plasma protein was increased (with high levels of albumin and globulin for males and only globulin for females) and associated albumin/globulin ratio was low for both sexes given 1000 mg/kg/day compared with Controls. Cholesterol was increased in both sexes given 1000 mg/kg/day, compared with Controls. Coupled with the microscopic findings in the liver of high dose animals, these changes in this group were considered to be test item-related. Females given 1000 mg/kg/day also showed increase in inorganic phosphorus and triglyceride concentration. Plasma chloride was slightly lower for both sexes given 1000 mg/kg/day. There were no test item-related macroscopic findings. Group mean absolute and adjusted liver weights were notably higher than Controls for both sexes given 1000 mg/kg/day (39% and 41% for males, and 45% and 37% for females, respectively) and slightly higher for males given 150 mg/kg/day (11%). Microscopic evaluation indicated hepatic centrilobular hepatocyte hypertrophy in animals given 1000 mg/kg/day. Group mean absolute and adjusted thyroid weights were higher than Controls for all treated males (21% to 35%). Group mean absolute and adjusted thyroid weights were also higher for females given 1000 mg/kg/day (13% and 4%). Microscopic evaluation indicated follicular cell hypertrophy in the thyroids of males and females given 1000 mg/kg/day. Group mean adjusted adrenal weights were higher than the Control mean for females given 1000 mg/kg/day and lower for females given 150 mg/kg/day. However, vacuolation in the zona fasciculata and the zona glomerulosa were observed in the adrenals of males only, given 1000 mg/kg/day. Hypertrophy/vacuolation of the interstitial gland was observed in the ovary in females given 1000 mg/kg/day; there were no changes to ovarian weights.

The test substance administered once daily by oral gavage to the rat at doses of 0, 15, 150 or 1000 mg/kg/day was tolerated for at least 90 days, but elicited test-item related ovarian changes in females and adrenal changes in males at 1000 mg/kg/day. In the absence of any functional investigations the adverse nature of these changes remains equivocal. In addition, significant increases in liver and thyroid weights in the presence of histopathological correlates were observed in males and females at this dose level. The No Observed Adverse Effect Level (NOAEL) for both sexes was considered to be 150 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

150 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The key study was performed in line with GLP and an accepted standardised guideline with a high standard of reporting. The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality as outlined in Klimisch (1997) and considered suitable for assessment as an accurate reflection of the test substance.

System:

endocrine system

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

Sub-chronic repeated dose toxicity

The oral repeated dose toxicity of the substance to the rat was investigated in a sub-chronic GLP study according to the OECD TG 408. Groups of 10 male and 10 female Crl:WI(Han) rats were dosed with 0 (vehicle), 15, 150 or 1000 mg/kg bw/day test substance, once daily by gavage, for at least 90 days, until the day before necropsy.

There were no deaths during the study. Excessive salivation was seen after dosing on a number of occasions throughout the treatment period in all dose groups, and the number and frequency of these occasions increased in a dose related manner. Yellow stained bedding was observed in cages for males given 150 mg/kg/day and both sexes given 1000 mg/kg/day. Throughout the study females given 1000 mg/kg/day consistently gained more weight than Controls. Their cumulative mean body weight gains and mean body weights at the end of the dosing period were 30% and 10% higher, respectively, than the Control values. There were no effects on body weight in males at 1000 mg/kg/day or in either sex at 15 or 150 mg/kg/day. A slight increase (+9%) in mean food intake was observed for females given 1000 mg/kg/day during the study. There was no notable effect on food intake during the study for males given any dose level and females given 15 or 150 mg/kg/day.

There were no test item-related effects on functional observations or on sensorimotor responses to visual, acoustic, tactile or proprioceptive stimuli at any dose level. All treated females were more active and travelled a greater distance than Controls for the first and second 10 minutes of motor activity monitoring, which was more notable for animals given 1000 mg/kg/day but with no differences from Controls for subsequent intervals. Forelimb grip strength was reduced for males given 1000 mg/kg/day, but their hind limb grip strength was similar to Controls. There were no effects on grip strength for females given 1000 mg/kg/day or either sex at any other dose level.

There were no ocular changes related to test substance administration. At 1000 mg/kg/day females had a statistically significant increase in white blood cells count due to increase in neutrophils and monocytes. Males had a statistically significant increase in platelets and reduced prothrombin time, compared with Controls. Bile acids were notably increased for females given 1000 mg/kg/day. Activity of aspartate aminotransferase was lower for both sexes given 1000 mg/kg/day and for females given 15 or 150 mg/kg/day. Males given 1000 mg/kg/day also had low alkaline phosphatase activity when compared with Controls. Group mean total plasma protein was increased (with high levels of albumin and globulin for males and only globulin for females) and associated albumin/globulin ratio was low for both sexes given 1000 mg/kg/day compared with Controls. Cholesterol was increased in both sexes given 1000 mg/kg/day, compared with Controls. Coupled with the microscopic findings in the liver of high dose animals, these changes in this group were considered to be test item-related. Females given 1000 mg/kg/day also showed increase in inorganic phosphorus and triglyceride concentration. Plasma chloride was slightly lower for both sexes given 1000 mg/kg/day.

There were no test item-related macroscopic findings. Group mean absolute and adjusted liver weights were notably higher than Controls for both sexes given 1000 mg/kg/day (39% and 41% for males, and 45% and 37% for females, respectively) and slightly higher for males given 150 mg/kg/day (11%). Microscopic evaluation indicated hepatic centrilobular hepatocyte hypertrophy in animals given 1000 mg/kg/day. Group mean absolute and adjusted thyroid weights were higher than Controls for all treated males (21% to 35%). Group mean absolute and adjusted thyroid weights were also higher for females given 1000 mg/kg/day (13% and 4%). Microscopic evaluation indicated follicular cell hypertrophy in the thyroids of males and females given 1000 mg/kg/day. Group mean adjusted adrenal weights were higher than the Control mean for females given 1000 mg/kg/day and lower for females given 150 mg/kg/day. Vacuolation in the zona fasciculata and the zona glomerulosa were observed in the adrenals of males given 1000 mg/kg/day. Hypertrophy/vacuolation of the interstitial gland was observed in the ovary in females given 1000 mg/kg/day; there were no changes to ovarian weights.

The test substance administered once daily by oral gavage to the rat at doses of 0, 15, 150 or 1000 mg/kg bw/day was tolerated for at least 90 days, but elicited test-item related ovarian changes in females and adrenal changes in males at 1000 mg/kg/day. In the absence of any functional investigations the adverse nature of these changes remains equivocal. In addition, significant increases in liver and thyroid weights in the presence of histopathological correlates were observed in males and females at this dose level. The No Observed Adverse Effect Level (NOAEL) for both sexes was considered to be 150 mg/kg/day.

Sub-acute repeated dose toxicity

The findings in the key study were supported by observations in a range finding and a 28-day oral repeated dose toxicity study in the rat. A 14 day range-finding study was performed to establish a top dose for a subsequent 28 day oral toxicity study. Four groups of two male and two female rats were dosed with 0, 250, 500 or 1000 mg/kg bw/day for a period of 14 days. Clinical observations, bodyweight and food consumption were recorded daily. At termination, a macroscopic examination post mortem was carried out which included excision and opening of the stomach to assess any possible irritant effects of the dosing preparation. There were no treatment-related clinical observations, no effects on bodyweight or food consumption and no macroscopic findings post mortem. A dose level of 1000 mg/kg bw/day was determined to be a suitable high dose level for the subsequent 28 day study. The chemical stability of the test material was determined to be satisfactory.

In a 28-day study, the oral repeat dose toxicity of the test material was determined in accordance with the standardised OECD TG 407. Groups of five male and five female rats were dosed orally by gavage with 0 (control), 15, 150 or 1000 mg/kg bw/day of the test material for 28 consecutive days.

At a dose level of 1000 mg/kg/day there were no adverse effects on bodyweights, food consumption or functional observations. Liver weights were increased in males and females receiving 1000 or 150 mg/kg/day. There were no treatment-related microscopic findings. Oral administration of 1000 mg/kg/day of the test material for 28 consecutive days resulted in toxicity as evidenced by marked increases in liver weight in both sexes. At this dose level small changes in certain haematology parameters, minor changes in cholesterol and minor changes in the activities of alkaline phosphatase and alanine aminotransferase were also noted. In animals receiving 150 mg/kg/day of test material, there were small increases in liver weight which were considered too small to be of toxicological significance in the absence of other indications of liver toxicity. Minimal difference in a small number of clinical pathology parameters were also noted at this dose level. The NOAEL for this study was determined to be 150 mg/kg/day.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:  Study selected is a 90 day study performed in line with GLP and standardised guidelines; supporting studies are a 28-day and a 14 day range-finding study.

Repeated dose toxicity: via oral route - systemic effects (target organ) - increase in liver and thyroid weights with associated histopathological changes

Justification for classification or non-classification

In accordance with the suggested criteria for classification as defined in Regulation (EC) No. 1272/2008, Annex I, 3.9.2.9, the test material does not require classification for specific target organ toxicity, repeated dose. The effects observed at the 150 mg/kg bw/day dose are not considered to be toxicologically significant and do not indicate any signs of organ dysfunction; the effects observed at 1000 mg/kg/day included test-item related ovarian changes in females and adrenal changes in males at 1000 mg/kg/day. In the absence of any functional investigations the adverse nature of these changes remains equivocal. In addition, significant increases in liver and thyroid weights in the presence of histopathological correlates were observed in males and females at this dose level.. For an oral subchronic study in the rat, the suggested guidance value for effects leading to classification is ≤100mg/kg bw/day, and with an NOAEL of 150 mg/kg bw/day no classification is warranted.