[No public or meaningful name is available]

Endpoint summary

Description of key information

Oral 28-day dietary toxicity with target substance containing 35% active ingredient was tested in rats at systemic dose levels of 0.05, 0.25 and 1.25% in the diet (as solids), corresponding to 48, 254 and 1225 mg act.ingr./kg bw/day. There were no adverse effects observed, therefore the dose of 1225 mg/kg bw/day can be considered as NOAEL.
Supporting data for subacute toxicity were available from an oral (gavage) OECD 422 study in rats with read across substance EC 939 -637 -2 at doses of 100, 300 and 1000 mg/kg bw/day. No relevant effects were observed at 100 and 300 mg/kg bw. At the dose of 1000 mg/kg bw, decreased body weight, increased serum ALAT and decreased serum albumin were observed as systemic changes, wheras macrosopic and microsopic stomach changes were observed as local changes, the latter wihtout relevance to human. NOAEL for paternal/maternal toxicity was 300 mg/kg bw/day.

Oral 90-day dietary toxicity studies with target substance (containing 35% active ingredient) were performed in rats at systemic dose levels of 0.5, 2 and 8 (reduced to 4) g act. ingr./kg bw/day and in dogs at 0.062, 0.250 and 1 g act. ingr. /kg bw/day. In rats, 2 and 8 (4) g act.ingr./kg bw/day were toxic, as demonstrated by decreased body weight and food consumption, serum changes, organ-to-body weight decreases and renal disease; the dose of 500 mg/kg bw/day can be considered as NOAEL. The dog study was considered to be less appropriate as the animals were very young and sensitive to gastro-intestinal irritation, although the study did not show systemic toxicity up to 1000 mg/kg bw/day.

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1976

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was conducted non-GLP, however it was conducted state of the art at the time of conduct. Although the animals were young at start of dosing, the study is considered relevant, reliable and adequate.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, Mass.
- Age at study initiation: weanlings
- Weight at study initiation: 50-79 g
- Fasting period before study: No data
- Housing: individual drawer-type wire mesh cages
- Diet : Charles River, Rat, Mouse and Hamster Meal.
- Water: ad libitum
- Acclimation period: Not provided

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24.4°C
- Humidity (%):45-65%
- Air changes (per hr):10
- Photoperiod (hrs dark / hrs light):12/12

Route of administration:

oral: feed

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: 0; 0.50; 2.00; 8.00 (four weeks) and 4.00 g/kg body weight/day for the remaining nine weeks
The diet was prepared by mixing, on a weigth basis, the compound with the basic diet in a Patterson-Kelley twin shell blender, equiped with a high speed mixing bar having slanted blades. Calculations were based on the weight of the animals for the second preceding week. The dosages express active compound, based on the 35% solid concentration of the sample.

DIET PREPARATION
- Rate of preparation of diet (frequency):Fresh diets were prepared each week
- Mixing appropriate amounts with (Type of food): Charles River, Rat, Mouse and Hamster Meal.
- Storage temperature of food: Not provided

VEHICLE: No Vehicle

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0; 0.50; 2.00; 8.00 (four weeks) and 4.00 g/kg body weight/day for the remaining nine weeks
Basis:
nominal in diet

No. of animals per sex per dose:

20

Control animals:

yes, plain diet

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
- Time schedule:daily observations were made and any abnormal conditions recorded.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined : Yes, weekly
and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at six weeks and at the termination of the study
- Anesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 5 rats of each sex (=10) from each group (4 groups)>40 rats
- Following parameters were examined:
Hematocrit Percentage
Hemoglobin Concentration
Total Leukocyte Count
Differential Leukocyte Count
RBC Morphology


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the conclusion of the experiment when possible
- Animals fasted: No data
- How many animals: the same rats as those used for hematology at the conclusion of the experiment= 40
- Following parameters were examined:
Serum Glucose Concentration
Blood Urea Nitrogen (BUN) Concentration
Serum Glutamic Oxaloacetic Transaminase (SGOT)
Serum Glutamic-Pyruvic Transaminase (SGPT)
Gamma Glutamyl Transpeptidase (GGTP)

URINALYSIS: Yes
- Time schedule for collection of urine: Twenty-four hour urine specimens were collected from 10 rats/group at six weeks and prior to sacrifice.
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Following parameters were examined:
Color
Appearance
Reaction(pH)
Specific Gravity
Protein
Sugar
WBC/h.p.f.
RBC/h.p.f.

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes , all rats underwent a thorough gross necropsy. Rats which had positive gross findings had either complete or partial (major organ) histology examination performed.
HISTOPATHOLOGY: Yes

Statistics:

Numerical data were treated statistically (student t test)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Two high dose rats died during the course of the experiment; male rat #5105 of acute hemorraghic prostatitis on the 27th day of the test, and female rat #5090 of hydrophilic degeneration of the liver and kidney on the 89th day.

Mortality:

mortality observed, treatment-related

Description (incidence):

Two high dose rats died during the course of the experiment; male rat #5105 of acute hemorraghic prostatitis on the 27th day of the test, and female rat #5090 of hydrophilic degeneration of the liver and kidney on the 89th day.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

[Trade name] depressed significantly the rate of body weight gain in the mid dose and high dose rats under the conditions of this experiment. This depression appeared more pronounced in the male than in the female rats.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

During weeks four through nine the feed consumption of the high dose male animals was significantly depressed. The other groups showed only an occasional significant depression.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

The feed efficiency of the mid dose and high dose test groups is decidedly different from that of the control group, more so in the male than in the female rats.

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Some rats in all levels exhibited slightly elevated SGOT values and 4 high dose rats exhibited elevated SGPT levels. Histologically, however, there was no basis for these elevations.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Some of the rats in the mid dose and high dose group displayed hematuria to a greater or lesser degree. Their urinary system was not remarkably changed histologically. Only one rat, #5103, high dose group, displayed hematuria and moderate acute cystitis.

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Decrease in thyroid weight in males of high dose group; decrease in adrenal and gonadal weight in high dose groups; decrease in pituitary weight in females of high dose group; decrease in brain weight in males of mid dose and high dose group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Rats of various groups had chronic respiratory disease. Two male rats had lower urinary tract pathology, 1 control rat had a pulmonary granuloma, 1 high dose rat had hydrophilic degeneration of liver and kidneys, 1 high dose rat had hepatic lipidosis.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Rats of various groups had chronic respiratory disease. Two male rats had lower urinary tract pathology, 1 control rat had a pulmonary granuloma, 1 high dose rat had hydrophilic degeneration of liver and kidneys, 1 high dose rat had hepatic lipidosis.

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
Due to decrease in body weight and feed consumption in the high dose group (8.00 g/kg/day) the level was lowered to 4.00 g/kg/day for weeks five through the termination of the study. Two high dose rats died during the course of the experiment; male rat #5105 of acute hemorraghic prostatitis on the 27th day of the test, and female rat #5090 of hydrophilic degeneration of the liver and kidney on the 89th day.

BODY WEIGHT AND WEIGHT GAIN
[Trade name] depressed significantly the rate of body weight gain in the mid dose and high dose rats under the conditions of this experiment. This depression appeared more pronounced in the male than in the female rats.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
During weeks four through nine the feed consumption of the high dose male animals was significantly depressed. The other groups showed only an occasional significant depression.

FOOD EFFICIENCY
The feed efficiency of the mid dose and high dose test groups was decidedly different from that of the control group, more so in the male than in the female rats.

HAEMATOLOGY
All rats had normal hematocrit concentrations for the duration of the experiment
All rats had normal hemoglobin concentrations for the duration of the experiment
Rat #5097 (4.00 g/kg/day group) had a WBC of 35000 cells/mL at six weeks but by three months its WBC was normal. The rest of the rats had normal white cell counts at all times examined.
All rats examined at six and 13 weeks displayed lymphocytosis.
The RBC’s of all animals were morphologically normal at all times.

CLINICAL CHEMISTRY
All rats had normal blood glucose values when examined.
All rats examined had normal BUN values.
Some rats in all levels exhibited slightly elevated SGOT values. Histologically, however, there was no basis for these elevations.
Four high dose rats exhibited elevated SGPT levels. Histologically, however, there was no basis for these elevations.
All rats examined had normal GGPT values.

URINALYSIS
Some of the rats in the mid dose and high dose group displayed hematuria to a greater or lesser degree. Their urinary system, however, was not remarkable histologically. Only one rat, #5103, high dose group, displayed hematuria and moderate acute cystitis

ORGAN WEIGHTS
Cfr. Table 3 and 4 (absolute & relative organ weights)

GROSS PATHOLOGY
Rats of various groups had chronic respiratory disease. Two male rats had lower urinary tract pathology, 1 control rat had a pulmonary granuloma, 1 high dose rat hydrophilic degeneration of liver and kidneys, 1 high dose rat hepatic lipidosis.

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: overall effects

Critical effects observed:

not specified

Table 1. Body weight data for rats receiving [Trade name] for 13 weeks

Dose levels (g/kg).	Mean male Body Weight (g)		Weight Gain (g)	Mean female Body Weight (g)		Weight Gain (g)
	Initial	Terminal		Initial	Terminal
0	68.4	509.0	440.6	60.0	289.3	229.3
0.50	68.3	505.6	437.3	60.1	276.7	216.6
2.00	68.4	434.5*	366.1	60.1	249.9*	189.8
8.00 (4.00)	68.2	269.8*	192.6	59.8	189.1*	129.3

* Statistically significant from control at P=0.05.

Table 2. Feed consumption for rats receiving [Trade name] for 13 weeks (g/rat/week)

Week on diet	Males				Females
	Dose levels (g/kg)				Dose levels (g/kg)
	0	0.50	2.00	8.00 (4.00)	0	0.50	2.00	8.00 (4.00)
1	103.8	97.2	112.5	101.5	83.3	90.3	86.8	84.0
2	124.8	119.0	133.5	137.0	103.5	116.5	106.3	115.3
3	157.3	157.8	162.8	163.0	134.8	144.3	150.5*	156.5*
4	157.3	135.0*	132.5*	186.1*	137.5	123.8	125.5	122.3
5	199.0	189.0	195.3	152.1*	158.3	138.0*	140.0*	150.8
6	185.7	185.7	190.1	162.8	145.0	159.2	168.8*	138.0
7	184.8	183.0	188.0	206.6*	130.8	131.0	137.1	170.8*
8	185.0	186.0	183.0	196.1*	150.3	138.5	152.8	164.3
9	175.3	178.5	184.3	194.2*	149.5	139.8	136.5*	166.0
10	181.0	179.5	184.0	176.3	134.8	129.3	138.8	150.8
11	182.5	191.0	183.7	177.1	147.5	153.2	143.2	164.5
12	179.0	178.4	176.3	182.4	160.0	145.0	158.3	172.8
13	190.0	188.9	177.0	160.5*	142.5	126.8	131.0	167.1
Mean	169.7	166.8	169.5	168.9	136.8	133.5	136.6	147.9

* Statistically significant from control at P=0.05.

Table 3. Summary of Mean Body Weights and Organ Weights on rats receiving [Trade name] in their diet for 13 weeks

Sex	Dose Level. g/kg	Terminal Body Weight    (g)	Organ Weights
			Thyroids	Heart	Liver		Adrenals	Kidneys	Gonads	Pituitary	Brain
Male	0	503.9	0.034	1.262	20.390		0.060	3.527	3.361	0.013	2.216
	0.50	503.7	0.031	1.274	22.540		0.060	3.566	3.491	0.013	2.107
	2.00	443.3*	0.033	1.158	15.884*		0.064	2.984*	3.179	0.014	2.084*
	8.00(4)	286.9*	0.025*	0.809*	9.890*		0.046*	1.926*	2.709*	0.010	1.906*
Female	0	301.6	0.033	0.879		11.326	0.085	1.966	0.152	0.017	1.969
	0.50	280.9	0.034	0.799		9.759*	0.080	1.900	0.169	0.017	1.969
	2.00	254.3*	0.031	0.755*		10.529	0.077	1.912	0.153	0.017	1.897
	8.00(4)	200.2*	0.032	0.636*		8.316*	0.061*	1.582*	0.101*	0.012*	1.938

*Statistically significant from control at P=0.05

Table 4. Organ-to-body weight ratios for male and female beagle dogs receiving [Trade name] in their diet for 13 week

	Dose Levels g/kg	Terminal Body Weight    (g)	(Grams of organ weight/kilograms of body weight)
Sex			Thyroids	Heart	Liver	Adrenals	Kidneys	Gonads	Pituitary	Brain
Male	0	503.9	0.067	2.504	40.464	0.119	6.999	6.670	0.026	4.398
	0.50	503.7	0.073	2.529	44.749	0.119	7.080	6.931	0.026	4.183
	2.00	443.3*	0.074	2.612	35.831	0.144	6.731	7.171	0.032	4.701
	8.00(4)	286.9*	0.087	2.820	34.472	0.160	6.713	9.442	0.035	6.643
Female	0	301.6	0.109	2.914	37.553	0.282	6.519	0.504	0.056	6.529
	0.50	280.9	0.121	2.844	34.742	0.285	6.764	0.602	0.061	7.010
	2.00	254.3*	0.122	2.969	41.404	0.303	7.519	0.602	0.067	7.460
	8.00(4)	200.2*	0.160	3.177	41.538	0.305	7.902	0.504	0.060	9.680

*Statistically significant from control at P=0.05.

 

 

Conclusions:

These studies have shown that the test item , when fed to random bred albino rats under the conditions of this experiment, decreased the rate of body weight gain, feed consumption and food efficiency at the mid dose and high dose levels, and increased SGOT and SGPT at the high doses. Further hematuria was seen in mid and high dosed rats, various organ weights were decreased and lower urinary tract pathology was seen in 2 high dosed rats.It thus appeared that the no effect level in the rat is below 0.50 g/kg/day, however 0.50g act.ingr./kg bw/day can be considered as NOAEL.

Executive summary:

Sprague-Dawley albino rats, 80 of each sex, were fed a control diet, or 0.50, 2.00 or 8.00 g/kg /day of test item, containing 35.8% active ingredient, mixed in the diet. The high dose was reduced to 4.00 g/kg/day of test item mixed in the diet for weeks five through to termination. The rats were carefully observed for the duration of the experiment and pertinent hematological and biochemical parameters were conducted while the experiment was in progress. At the conclusion of the experiment all rats were necropsied and all organs and tissues were examined histologically from ten rats (five male and five female) from the control and high dose groups. These studies have shown that the test item, when fed to random bred albino rats under the conditions of this experiment, decreased body weight gain, feed consumption and food efficiency at the mid dose and high dose levels and increased SGOT and SGPT at the high doses. Further hematuria was seen in the mid and high dose rats, various organ weights were decreased (e.g. decrease in adrenal and gonadal weight in high dose groups; decrease in pituitary weight in females of high dose group) and lower urinary tract pathology was seen in 2 high dosed rats. It thus appeared that the no effect level in the rat is below 0.50 g/kg/day, however 0.5g act.ingr./kg bw/day can be considered as NOAEL.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

High quality (Klimish 2)

System:

other: gastrointestinal (hepatobiliary) & urinary tract

Organ:

kidney

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

Mode of Action Analysis / Human Relevance Framework

In the absence of any evidence for species specific effects or modes of action the effects observed in animals are regarded as relevant for humans.

Additional information

Subacute toxicity

- In a subacute 28 day toxicity study, liquid target substance containing 35% act.ingr. of registered test item was added to the basic diet of 4 groups of 10 young male albino rats at dietary levels of 0 (control), 0.05, 0.25 and 1.25% active ingredient (Tusing, 1955). There were no mortalities and no adverse effects on growth or food consumption and autopsies revealed no gross pathology that could be attributed to the dietary feeding of the compound. A NOAEL of 1250 mg act.ingr./kg bw/day was determined, however not all parameters were studied (e.g. haematology, clinical biochemistry and histopathology were missing). The study was considered to be supporting, and no further 28 -day study was needed based on availability of 90-day toxicity data.

- A supporting subacute toxicity study with read across substance EC 939 -637 -2 was available from an oral combined repeated dose and reproduction/development screening study according to OECD guideline 422 (Hansen, 2013). The test item was administered orally by gavage to rats with a liquid formulation containing 41.5% active ingredient at dose levels of 100, 300 and 1000 mg/kg bw/day for at least 28 days in male rats and at least 39 days in females. No test item-related premature death was noted in any treatment group. No signs of clinical toxicity were noted for the male and female rats of the low and intermediate dose groups (100 and 300 mg/kg bw/day), whereas slightly increased salivation was noted in one male rat at 1000 mg/kg bw/day. No observational and functional neurological findings were seen up to the highest dose group. A slight reduction in body weight was noted for the male and female rats dosed at 1000 mg/kg bw/day. The laboratory examinations revealed an increased serum ALAT activity for the male and female rats dosed at 1000 mg/kg bw/day, and a decreased serum albumin concentration for the male rats of the high dose group. No test item-related changes were noted during the macroscopic inspection at autopsy with the exception of changes in the stomach from 2 male animals from the high dose group. Microscopic examination revealed the occurrence of squamous cell hyperplasia in the non-glandular mucosa of the forestomach and acute inflammation in the male and female rats of the high dose group. These changes are considered to be local, and not relevant for humans as humans lack a forestomach. NOAEL for paternal/maternal toxicity was 300 mg/kg bw/day.

- Further, a 14-day dose-range-finding was recently conducted with the target substance containing 34.4% active ingredient (Hansen, 2013b). Male and female rats were treated orally with 100, 300 or 1000 mg act. ing. /kg bw/day. No rats died prematurely nor revealed any test item-related changes in behaviour, external appearance or faeces. No changes in body weight and body weight gain, food and drinking water consumption or for relative and absolute organ weights were noted at any of the tested dose levels. Macroscopic examination revealed no test item-related changes at any of the tested dose levels. The study confirmed the results of the existing subacute toxicity study, therefore a new repeated dose toxicity study was not deemed necessary.

- In conclusion, subacute toxicity testing was performed, although these were supporting data. As 90-day studies were available, no further subacute testing was deemed necessary.

 

Subchronic toxicity

- In a key subchronic repeated dose toxicity study with target substance, 160 Sprague-Dawley albino rats, 80 of each sex, were fed a control diet, or 0.50, 2.00 or 8.00 g/kg /day of test item mixed in the diet (Tegeris and Underwood, 1976a). The liquid test item contained 35.8% active ingredient, which was taken into account for the dosages. The high dose was reduced to 4.00 g/kg/day mixed in the diet for weeks five through to termination. The study showed decreased body weight gain, feed consumption and food efficiency at the mid dose and high dose levels and increased liver enzymes (SGOT and SGPT) at the high dose. Further hematuria was seen in the mid and high dose rats, various organ weights were decreased (e.g. decrease in adrenal and gonadal weight in high dose groups; decrease in pituitary weight in females of high dose group) and lower urinary tract pathology was seen in 2 high dosed rats. It thus appeared that the NOEL in the rat was below 0.50 g/kg/day, however 0.5g act.ingr./kg bw/day can be considered as NOAEL.

- In another subchronic repeated dose toxicity study, target substance containing 35.8% active ingredient was given in the diet to purebred beagle dogs for ninety days (Tegeris and Underwood, 1976b). Thirty-two purebred beagle dogs, sixteen of each sex, with an average age of three to four months, were fed the control diet or 0.062, 0.250 or 1.000 g act.ingr. /kg bw/day thoroughly mixed in the diet. The dogs were carefully observed for the duration of the experiment and several hematological and biochemical parameters were conducted while the experiment was in progress. At the conclusion of the experiment all dogs were necropsied and all organs and tissues were examined histologically. These studies have shown that the test substance interfered with the average daily feed consumption of the mid-dose female and high dose test dogs and decreased the rate of bodyweight gain of the high dose test dogs when fed to them under the conditions of this experiment. Otherwise it was harmless up to 1 g/kg bw/day; a NOAEL of 0.250 g/kg bw/day can be considered.

- In conclusion, NOAEL-level of 500 mg/kg bw was considered as the most appropriate value for further risk characterisation.

 

Conclusion

- For risk characterisation, NOAEL of 500 mg/kg bw in the 90 -day rat repeated dose toxicity study with registered substance was selected.

- Further information supporting the safety of the test substance is provided in the read across justification for the Mono-ester subgroup, (justification with data matrix separately attached in Section 13).

Justification for classification or non-classification

Available data suggest values that warrant no classification for repeated dose toxicity under Regulation 1272/2008 (CLP) nor under Directive 67/548/EC (DSD).