A mixture of branched and linear C7-C9 alkyl 3-[3-(2H-benzotriazol-2-yl)-5-(1,1-dimethylethyl)-4-hydroxyphenyl]propionates

Endpoint summary

Description of key information

28 day study: NOAEL (male/female) = 50 mg/kg bw (OECD Guideline Study, CIBA, 1991)
                         NOEL (male/female) = 2 mg/kg bw
                         LOAEL (male/female) = 500 mg/kg bw
                         
                        

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:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From Sep. 23, 1987 to Dec. 12, 1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study conducted according to GLP. Histopathological examinations were not performed for all tissues. In accordance to ECHA guidance, the use as read-across reduces the reliability to 2. The study itself is valid without restrictions.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1981 guideline followed, reliability scoring based on 1998 guideline

Deviations:

yes

Remarks:

histopathological examinations were not performed for all tissues

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain as stated in the report: Sprague Dawley Crl CD (SD) BR
- Source: Centre d'Elevage Charles River (76140 Saint-Aubin-les-Elbeuf, France)
- Age at study initiation: 6 weeks
- Weight at study initiation: approx. 158g females and approx. 200 g males
- Housing: 2 same-sex animals from the same group per cage (wire-mesh, 43 x 21.5 x 18 cm); cages were placed in order vertically on racks and racks were moved around in a clockwise manner fortnightly.
- Diet ad libitum: pellet diet (ref. A04 C, U.A.R., 913 Villemoisson0sur-Orge, France)
- Water ad libitum: tap water filtered with 0.22 micron F.G. Millipore filters
- Acclimation period: 11 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 50 ± 20
- Air changes (per hr): 13
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Dilutions of test article in polyethylene glycol were prepared and homogenized with a magnetic stirrer. The preparation was performed twice a week by C.I.T. Pharmacy according to the stability results obtained before the beginning of the study.

VEHICLE
Concentration in vehicle: 0.4, 1, 2, 10 mg/ml PEG
Amount of vehicle: 5 ml/kg body weight/day (amount of test substance given was individually adjusted every week to the most recent body weight)
Lot/batch no.: Batches Nos. 82202 and 87163

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the dosing solutions in polyethylene glycol 300 was checked before the beginning of the study on days 0, 1, 3, and 7. The concentrations achieved were examined on week 1, 4, 8 and 12 of the study by spectrophotometry at 301 nm. The substance was stable in polyethylene glycol under study conditions and the achieved concentrations were comparable to the nominal concentrations (the difference was always lower than 9 %).

Duration of treatment / exposure:

After 91 days 12 animals of control and 20 animals from the 10 mg/kg bw/d group were saved for a 30 day recovery period. Remaining males were sacrificed after 92 days and remaining females were sacrificed after 93 days of treatment.

Frequency of treatment:

once daily, 7 days per week

Remarks:

Doses / Concentrations:
2, 5, 10, 50 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

0 mg/kg bw/day - 16/sex
2 mg/kg bw/day - 10/sex
5 mg/kg bw/day - 10/sex
10 mg/kg bw/day - 20/sex
50 mg/kg bw/day - 10/sex

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: not reported
- Rationale for animal assignment: random
- Details on satellite groups: after the 91-day treatment, the first surviving 6 males and 6 females from the control group and 10 males and 10 females from the 10 mg/kg bw/day group were saved for a 30-day recovery period.

Positive control:

Not applicable

Observations and examinations performed and frequency:

CLINICAL SIGNS AND MORTALITY
The animals were observed daily for clinical signs and twice daily for mortality.
BODY WEIGHT
The weight of each animal was recorded 7 days prior, on the first day of treatment and weekly during the treatment and the recovery period.
FOOD CONSUMPTION
The food consumption by the animals of each cage was recorded weekly during treatment and recovery.
OPHTHALMOSCOPIC EXAMINATION
The animals from the control and 50 mg/kg bw/day group were subjected to ophthalmoscopic examinations before the first treatment and in week 13.
HAEMATOLOGY AND CLINICAL CHEMISTRY
In week 13 and after recovery (week 17) blood was collected under light ether anesthesia from the orbital sinus in overnight fasted animals for haematology and clinical chemistry. Parameters given in table 1 were examined.

Sacrifice and pathology:

GROSS PATHOLOGY
All surviving animals were sacrificed 18 hours after the last administration and after an 18 h fasting period after end of the recovery period, respectively. Macroscopic examinations of all animals including those that died during the study were performed. Tissues listed in table 2 were preserved in 10 % buffered formalin.
ORGAN WEIGHTS
The following organs from all animals sacrificed at termination were weighed: Adrenals, kidneys, liver, ovaries, spleen, testes, seminal vesicles and prostate. Body and organ weights were not recorded at necropsy in the animals that were found dead.
HISTOPATHOLOGY
Histopathological examinations were performed on organs given in Table 2 of all animals from the control and 50 mg/kg bw/day groups. Samples were embedded in paraplast and were stained with hemalum and eosin. All tissues not required for microscopic examinations were kept in fixative. At 2, 5 and 10 mg/kg bw/day all tissues with macroscopical abnormalities, lungs, liver, kidneys as well as seminal vesicles, prostate, testes and epididymides from the 10 mg/kg bw/d group were examined at the end of treatment. At the end of the recovery period all tissues with macroscopical abnormalities and liver, lungs, kidneys, seminal vesicles, prostate, testes and epididymides were examined in all animals.

Statistics:

A normal distribution of values in the samples was checked using KOLMOGOROV-SMIRNOV's test. In the case of an abnormal distribution, this test was performed after logarithmic transformation of the values. If a significant heterogeneity persisted after logarithmic transformation, the analysis of variances was not performed. A comparison between the treated groups and the control group in order to prove a treatment related difference was made using MANN-WHITNEY's test.
In case of normal distribution of values according to the normal law, an analysis of variances was made using BARTLETT's test (more than two samples) or FISHER's test (two samples).
A comparison between the treated and control groups in order to prove a treatment related difference was made using: DUNNETT's test, if no significant heterogeneity of the variances was established; MANN-WHITNEY's test, in the case of significant heterogeneity of the variance.
A slight difference may appear from time to time with the rounded number of the mean and standard deviation between the individual values and the summary section of the statistical tests. These differences are due to the use of different calculators in data processing, which are not processed the same way in the central memory. These differences, when they occur, are always seen in the last decimal and do not affect the scientific value of the results.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

transient reduced body weight in males of the high dose group.

Food consumption and compound intake (if feeding study):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

ALP, albumin, decrease in alpha-1, alpha-2, beta and/or gamma globulins

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

liver, increased at 5 mg/kg bw and above

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

dose dependently enlarged livers in males

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

slight liver hypertrophy in males of the high dose group.

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS
No treatment related clinical signs were observed. Clinical signs like piloerection, loss of body weight, chromorhinorrhea, chromodacryorrhea, round back, loud breathing or dyspnea, and decrease in activity were seen sporadically in animals of all groups. They were not considered to be of toxicological significance. Other clinical signs like cutaneous lesion or hair loss were considered to be usually observed in the laboratory rat.
MORTALITY
There were no deaths during the recovery period. Mortality during the treatment period was as follows:
0 mg/kg bw/day - 4/16 males (25%) and 1/16 females (6%)
2 mg/kg bw/day - 0/10 males (0%) and 1/10 females (10%)
5 mg/kg bw/day - 1/10 males (10%) and 0/10 females (0%)
10 mg/kg bw/day - 2/20 males (10%) and 0/20 females (0%)
50 mg/kg bw/day - 0/10 males (0%) and 1/10 females (10%)
The factors contributing to mortality were, in most cases, the same in controls and treated animals. Two deaths were due to experimental mistakes, and, in males, the mortality rate was higher in the controls than in the treated groups. Therefore, it was not considered to be of biological significance.

BODY WEIGHT AND WEIGHT GAIN
The male body weight was slightly but significantly decreased from Week 6 in males at 50 mg/kg bw/day (ranging from 94 to 84 % of control). This effect was not significant at week 13 (94%) but was considered to be treatment related. Other effects on body weight were either not dose dependent or did not reach statistical significance and were therefore not considered treatment related.

FOOD CONSUMPTION
Food consumption of treated males was similar to control males without any statistically significant difference (except in week 4 in the 50 mg/kg/day group). The food consumption of treated females was also similar to control females. The difference was occasionally statistically significant in week 7 in the 5 mg/kg/day group. These variations were not considered to be of biological significance.

OPHTHALMOSCOPIC EXAMINATION
Upon ophthalmoscopic examination no test article related abnormality was noted. However, minor changes like cornea vacuolization and persistence of hyaloid vessel were seen at 0 and 50 mg/kg bw/day, before the start of treatment and at week 13. Since these findings are not uncommon in the laboratory rat (Heywood R, Laboratory animals 7, 19-27, 1973) they were considered to be not treatment related.

HAEMATOLOGY
Findings like higher thrombocyte values in males in all treated groups, decreased mean haemoglobin content (males) and mean value of packed cell volume (male and female), lower quick time values, increased lymphocyte count or different white blood cell count (male and female) were seen. These findings were either within the historical controls, the significance was due to high individual values or changes were not dose dependent. Therefore, these findings were considered to be of no biological significance.
After recovery, no differences were observed in the 10 mg/kg bw/day group except for lower values in packed cell volume, mean cell volume, and mean cell haemoglobin, which were not considered to be of biological significance.

CLINICAL CHEMISTRY
Increased alkaline phosphatase activity was seen in males and females at 50 mg/kg bw/day (factor of 1.4 and 2.9 for females and males, respectively) and in males at 10 mg/kg bw/day (factor of 1.5). An increase in albumin levels was noted in males and females from the 10 and 50 mg/kg bw/day groups, with a decrease in alpha-1, alpha-2, beta and/or gamma globulins. These decreases were also noted in females from the 2 and/or 5 mg/kg bw/day groups. Further details are given in table 3. Although these changes were not accompanied by any organic lesions in the organ that could be the source, they were considered related to the administration of the test article. At the end of the recovery period, these abnormalities were totally reversible.
Other significant changes were:
Decreased sodium levels (142 and 142 mmol/l compared to 144 mmol/l at control) and chloride levels (105 and 106 compared to 109 mmol/l) in males at 10 and 50 mg/kg bw/d and increased levels of inorganic phosphorus and glucose in males at 50 mg/kg bw/d (2.8 versus 2.3 and 8.7 vs. 7.3 mmol/l at control, respectively). Males also had a lower bilirubin level (2 and 2 vs. 3 µmol/l at control) as well as increased albumin/globulin ratios (1.7 and 2.1 vs. 1.3 at control) and alkaline phosphatase activity (224 and 428 vs. 148 UI/l) at 10 and 50 mg/kg bw/d.
Further changes in females consisted of decreased calcium levels at 2, 5 10, 50 mg/kg bw/d (2.8, 2.7, 2.8, 2.7 vs. 2.9mmol/l at control), decreased total bilirubin level (2, 2, 2 vs. 3µmol/l at control) at 5, 10 and 50 mg/kg bw/day, increased albumin/globulin ratios at 2, 5, 10 and 50 mg/kg bw/d (2.1, 2.1, 2.2, 2.6 vs. 1.7 at control), increased alkaline phosphatase (ALP) activity at 50 mg/kg bw/d (108 vs. 78 UI/l at control), as well as decreased aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT) activity at 2, 5, 10, 50 mg/kg bw/d (54, 59, 62, 59 vs. 75 UI/l at control and 28, 29, 24 and 25 vs. 53 UI/l at control, respectively).
The decreases in ALAT and ASAT mean group values were due to individual high values in the control group. The individual values in the treated groups were comparable to those of the controls and to the normal historical values, therefore they were considered to be of no biological significance. Other changes seen in clinical biochemistry like decreased cholesterol in females at 10 mg/kg bw/d were not dose dependent.
All effects at 10 mg/kg bw/day were partially (beta-globulin, alpha-1-globulin) or fully (ALP activity, albumin, alpha-2-globulin) reversible.

GROSS PATHOLOGY
Macroscopic examination at the end of treatment revealed dose dependently enlarged livers in males (1/10, 7/10 and 10/10 at 5, 10, and 50 mg/kg bw/day). These findings were correlated with hepatic cell hypertrophy in 3/10 males from the 50 mg/kg bw/day group at the end of treatment. No abnormality was observed in females and in both sexes after the recovery period. The hepatotropic effect observed at all dose levels in males was considered to indicate an adaptive change of a functional nature.

ORGAN WEIGHTS
A dose dependent increase in the net and relative liver weight in males and females from all treated groups was noted at the end of the treatment period. These variations were correlated with enlarged livers and hepatic cell hypertrophy at 50 mg/kg bw/day. They were not present at the end of the recovery period. A decrease in the net adrenal weight and an increase in the relative kidney and testes weights were noted in males from the 50 mg/kg bw/day group. In the absence of any microscopic findings, these changes could be related to the decrease in body weight in these animals. Other findings like a significantly increased relative and absolute spleen weight at 5 mg/kg bw/day in females was not associated with histopathological findings and was considered to be of no biological significance.

HISTOPATHOLOGY
A minimal to slight hepatic cell hypertrophy was found in 3/10 males at 50 mg/kg bw/day. This change could be related to the administration of the test substance. The incidence, severity, and morphological characters of other microscopic changes like congestions of adrenal glands in one female each of the 0, 2 and 10 mg/kg bw/day group did not suggest a treatment relationship.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The substance affects the liver. As shown in separate mechanistic studies, it is a potent peroxisome proliferator.

Critical effects observed:

not specified

Table 3: Selected biochemical parameters

MALES after 13 week treatment
Dose mg/kg bw/day	Albumin		A1-Globulin		A2-Globulin		Beta-Globulin		G-Globulin
	%	g/l	%	g/l	%	g/l	%	g/l	%	g/l
0	56.3	42.5	9.7	7.3	5.8	4.3	18.4	13.9	9.9	7.4
2	58.8	44.4	7.8	6.0	4.7	3.6	18.2	13.8	10.4	7.8
5	59.9 *	45.7	8.2	6.3	4.8	3.6	16.8 *	12.9	10.3	7.9
10	62.7 **	47.5 **	7.4 *	5.6 *	5.0	3.9	16.0 **	12.1 **	9.0	6.8
50	67.7 **	52.5 **	6.5 **	5.0 **	3.8 **	2.9 **	14.0 **	10.8 **	8.2	6.3
MALES after recovery (17 weeks)
0	62.1	50.4	3.8	3.1	4.6	3.8	18.3	14.9	11.2	9.2
10	63.3	50.7	5.6 **	4.5 *	4.3	3.4	16.6 *	13.3 *	10.2	8.2
FEMALES after 13 week treatment
Dose mg/kg bw/day	Albumin		A1-Glubulin		A2-Globulin		Beta-Globulin		G-Globulin
	%	g/l	%	g/l	%	g/l	%	g/l	%	g/l
0	62.6	48.3	5.8	4.5	4.1	3.2	17.7	13.6	9.9	7.6
2	67.7 *	51.0	4.3	3.2 *	3.3	2.5	15.8	11.9 *	9.0	6.8
5	66.8 *	51.1	3.9 *	3.0 *	3.2	2.4 *	15.8	12.0 *	10.3	7.8
10	68.5 **	53.5 **	4.2 *	3.3 *	3.0 **	2.3 **	15.4 **	11.9 **	9.0	6.9
50	71.6 **	57.9 **	3.5 **	2.8 *	3.6	2.9	13.8 **	11.2 **	7.4 *	6.0 *
FEMALES after recovery (17 weeks)
0	67.9	55.8	3.5	2.9	3.4	2.8	15.5	12.7	9.7	7.9
10	69.4	56.9	3.9	3.2	3.1	2.5	13.6	11.1 *	10.1	8.2
*, p<0.05; **, p<0.01

Table 4: Mean body weights, absolute and relative mean organ weights (g and g/100g bw, respectively) after 13 weeks.

MALES
		Dose (mg/kg bw/d)
		0	2	5	10	50
Body weight		458	430	443	446	411 *
Sem. Ves.	Absolute	1.706	1.590	1.633	1.669	1.559
	relative	0.373	0.372	0.370	0.374	0.378
Testes	Absolute	3.539	3.488	3.511	3.539	3.540
	relative	0.774	0.816	0.796	0.795	0.865 *
Prostate	Absolute	1.456	1.457	1.411	1.385	1.328
	relative	0.319	0.338	0.319	0.310	0.322
Adrenal glands	Absolute	0.066	0.058	0.062	0.061	0.055 *
	relative	0.014	0.014	0.014	0.014	0.013
Liver	Absolute	12.355	12.744	14.659 *	16.390 **	22.882 **
	relative	2.691	2.960 *	3.320 **	3.674 **	5.549 **
Spleen	Absolute	0.959	0.817	0.981	0.856	0.830
	relative	0.209	0.189	0.223	0.192	0.201
Kidneys	Absolute	3.693	3.657	3.649	3.979	3.740
	relative	0.809	0.853	0.826	0.891	0.910 *
FEMALES
		Dose (mg/kg bw/d)
		0	2	5	10	50
Body weight		257	251	258	259	249
Ovaries	Absolute	0.117	0.117	0.119	0.126	0.133
	relative	0.046	0.046	0.046	0.049	0.054
Adrenal glands	Absolute	0.067	0.068	0.064	0.067	0.062
	relative	0.026	0.027	0.025	0.026	0.025
Liver	Absolute	7.315	7.476	8.407 *	8.374 *	10.694 **
	relative	2.838	2.974	3.268 **	3.243 **	4.292 **
Spleen	Absolute	0.557	0.622	0.697 *	0.610	0.573
	relative	0.217	0.248	0.272 *	0.237	0.231
Kidneys	Absolute	2.150	2.161	2.119	2.188	2.277
	relative	0.839	0.859	0.825	0.846	0.914
*, p<0.05; **,p<0.01

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

2 mg/kg bw/day

Study duration:

subchronic

Species:

rat

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

For the substance itself (EC no 407 -000 -3)

, a subacute gavage study in rats is available (Ciba 1991). A subchronic toxicity study was not performed since sufficient data from structurally related esters is available. Read-across for the 90 -study with EC 400 -830 -7 was approved by the EU member state competent authority. A data matrix with relevant properties and substances is shown below. The pattern observed for acute and repeated dose toxicity is sufficiently similar. The skin sensitizing properties of EC 400 -830 -7 are considered to be related to the polyethylene glycol chains that are unique to this substance. It may be prone to epoxide formation.

Table 1: Data matrix for physicochemical properties

			Hexane-1,6-dyl (3-(benzotriazol-5-tert-butyl-4- hydroxyphenyl)propionate)	3-(benzotriazol-5-tert-butyl-4- hydroxyphenyl) propionic acid	3-(benzotriazol-5-tert-butyl-4-hydroxyphenyl)propionic acid methyl ester
	EC 407-000-3	EC 400-830-7	CAS 84268-08-6	CAS 84268-36-0	CAS 84268-33-7 EC 400-820-2
Mol. weight	451.61	Mean MW monoesters: 637; mean MW diesters 975	760.94	339.4	335.4
Melting point	-29.4°C	-40°C	117 °C	195°C	120°C
Relative densitiy	1.08	1.17g/cm^3	1.21		1.24
Water solubility	0.018 mg/L (at 20°C)	1.74 mg/L (n6-Monoester), <0.2 mg/L (n6-diester) Surface-active	<0.5 mg/L		<0.05 mg/L
Fat solubility	96.6 g/100 g of fat (at 37°C)		1.61 g/100 g of fat
Log Pow  (calculated from solubilities)	9.2 (calculated)	4.6 and 5.9 (n6 Mono-and diesters)	11 (calculated)	4.19 (calculated)	4.48 (calculated)

Table 2: Data matrix for toxicological properties

	EC 407 -000 -3	EC 400 -830 -7	CAS  84268 -08 -6	CAS 84268 -36 -0	CAS 84268-33-7
Skin and eye irritation	Not irritating (K1)	Not irritating (K1)	Not irritating (K1)	Not irritating (K2)	Not irritating (K1)
skin sensitzation	Not sensitizing (K1)	Sensitizing (K1)	Not sensitizing (K1)		Not sensitizing (K1)
acute oral tox (LD50 in mg/kg bw)	No mortality at 2000 mg/kg bw (K1)	No mortality at 2000 mg/kg bw (K1)	No mortality at 5000 mg/kg bw (K2)	LD50 > 2000 mg/kg bw (K2)	No mortality at 5000 mg/kg bw (K2)
acute dermal tox (LD50 in mg/kg bw)	No mortality at 2000 mg/kg bw (K1)	No mortality at 2000 mg/kg bw (K1)	No mortality at 2000 mg/kg bw (K2)		No mortality at 2000 mg/kg bw (K2)
90-day gavage study (OECD 408)		NOEL = 2 mg/kg bw Peroxisome proliferation shown	NOEL = 2 mg/kg bw Peroxisome proliferation shown
28-day gavage study (OECD 407)	NOAEL = 50 mg/kg bw NOEL = 2 mg/kg bw	NOEL = 10 mg/kg bw	NOEL < 400 ppm (ca 33 mg/kg bw)	NOEL < 50 mg/kg bw
14-day studies				0, 10, 50 and 200 mg/kg bw, peroxisome proliferation shown	0, 10, 50 and 200 mg/kg bw, peroxisome proliferation shown
Ames test	Not mutagenic (K1)	Not mutagenic (4 strains, K2)	Not mutagenic (K1)	Not mutagenic (K1)	Not mutagenic ( 4 strains, K2)
Clastogenicity in vitro	Not clastogenic (K1)	Not clastogenic (K1)
Mutagenicity in mammalian cells in vitro
Clastogenic-ity in vivo (MN)	Not clastogenic (K1)	Not clastogenic (K1)	Not clastogenic (K2)		Not clastogenic (K2)
Mutagenicity in mammalian cells in vivo (UDS) OECD 482		Not mutagenic (K1)
One-generation study in rats (OECD 415)		NOEL = 2 mg/kg bw (50 and 100 mg/kg bw: reduced litter sizes, increased number of stillborn rats, increased pup mortality immediately after birth, and reduced birth weights.) Peroxisome proliferation shown in rat fetus
Teratogenicity in rats (OECD 414)		Not teratogenic
Teratogenicity in rabbits (OECD 414)		Not teratogenic
Toxikokinetic study available			yes		yes
Acute inhalation toxicity (OECD 403)		no mortality at 5.8 mg/L (aerosol, K1)

The following section summarizes the findings on the substance itself. The effects are consistent with those obtained after treatment with the related esters.

Subsequent to an oral range finding study, an oral subacute toxicity study (28 d) was conducted, according to OECD guideline 407 (CIBA, 904257). Groups of 20 male and 20 female rats were dosed orally via gavage over 28 days with the test substance at dose levels of 0, 2, 50 and 500 mg/kg bw/day (groups 1 to 4). In each dose group, 10 animals per sex and group were sacrificed at the end of the treatment period (experimental groups I (laboratory investigations, biochemistry, and histopathology) and III (neurology testing)), and 10 animals per sex and group were kept for a 4-week recovery period before sacrifice (experimental groups II (laboratory investigations, biochemistry, and histopathology) and IV (neurology testing)).

No treatment-related clinical symptoms and no signs of systemic toxicity were observed throughout the study. The mean body weight was reduced in the animals of the high dose group due to reduced food consumption. The neurological investigations (clinical signs, body temperature, reflex testing, grip-strength and land foot splay) and neurohistopathology did not reveal any neurotoxic effect.

Some animals of the two higher dose groups showed signs of a mild anaemia indicated by slightly lower erythrocyte, haemoglobin and hematocrit values. The reticulocyte counts and the platelet numbers were slightly enhanced.

The liver was found to be the main target organ: Absolute and relative liver weights were significantly increased in males of the 50 and 500 mg/kg bw/day and in females of the 500 mg/kg bw/day groups. Histopathology revealed a hypertrophy of the hepatocytes. The hypertrophy was accompanied with a minimal to moderate incidence of single cell or focal hepatocyte necrosis. The morphological effects seen on the liver correlated well with results from clinical chemistry. In the thyroid gland, a slight activation was noted. This slight hypertrophy of the thyroid follicular epithelium is considered to be a secondary effect to the liver hypertrophy. The effect on the thyroid is obviously due to a pituitary hormone-induced stimulation, which itself is the consequence of an increased elimination of plasma thyroxin by the stimulated liver.

Information on EC 400 -830 -7 is summarized below:

In a 7 day oral range finding study, 1000 mg/kg bw/d test article was administered daily via gavage to SD-rats (CIT 1986b). Mortality (1/10 animals), clinical signs, like piloerection and reduced activity, decreased bodyweight gain and a reduced food consumption compared to common values for animals of this age and strain were seen.

In a GLP-compliant subacute 28 day study conducted similar to OECD 407 doses of 10, 50, 200 and 1000 mg/kg bw/d were applied to SD-rats via gavage without recovery period (CIT 1986a). At 10 mg/kg bw/day a slight increase in absolute and relative male mean liver weight was the sole effect observed. No macroscopic or microscopic correlates were found. Therefore, 10 mg/kg bw/day was established as NOAEL. Due to increased absolute and relative liver weights as well as increased albumin level in both sexes, signs of liver necrosis in 1 animal and alopecia in females, the LOAEL was 50 mg/kg bw/day.

In the GLP-compliant oral subchronic 90 day toxicity study, conducted according to OECD guideline 408 (1981) with minor deviations doses of 2, 5, 10, and 50 mg/kg bw/d were administered by gavage to SD-rats (CIT 1988). Satellite groups of control and 10 mg/kg bw/d animals were allowed to recover for 4 weeks after the last treatment. Dose dependent hepatotropic effects were correlated with dose dependent enlargement of the liver (beginning at 5 mg/kg bw/day) and with hepatocyte hypertrophy at 50 mg/kg bw/day in males. In addition, some changes in plasma alkaline phosphatase activity and albumin and globulin concentrations were seen. Dose dependent and significant changes in liver weights were seen at 2, 5, 10 and 50 mg/kg bw/day (males and females, respectively).

Based on the results mentioned above the NOAEL of 2 mg/kg bw/d derived in the subchronic oral toxicity study was established as NOAEL for repeated dose oral toxicity.

However, several studies in rats are available, which confirm that the substance is a potent peroxisome proliferator in the liver after oral application (details see IUCLID-Chapter 7.9.3, specific investigations). The susceptibility to peroxisome proliferators is known to be a rodent specific phenomenon and therefore hepatotoxic effects seen in the repeated dose studies mentioned above are negligible to humans. Despite effects attributable to hepatotoxicity a slightly decreased body weight was seen in males (84-94% to control) at 50 mg/kg bw/d in the oral subchronic 90 day toxicity study solely.

All effects seen in this study have been shown to be reversible within the 4-week treatment-free period. From the effects found it is concluded that the NOAEL of this study is 50 mg/kg bw/day. The "no observable effect level" (NOEL) for the test article is defined as 2 mg/kg bodyweight for rats of either sex.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
GLP-compliant guideline Study

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver

Justification for classification or non-classification

The most prominent findings upon repeated dose toxicity were observed in the liver that are adaptive at the lower dose levels. Experimental data on structurally related esters shows that the acid part is a potent inducer of peroxisome proliferator in rats. Rats are known to be particularly sensitive to such effects and therefore, no classification for repeated-dose toxicity was included in the EU Annex I legal classification of EC 400 -830 -7.

Dangerous Substance Directive (67/548/EEC) (Annex I indox no. 607 -281 -00 -4):

The available studies are considered reliable and suitable for classification purposes under 67/548/EEC. In the subacute oral toxicity study, effects were non-adverse at 50 mg/kg bw and adverse at 500 mg/kg bw. No serious and irreversible effects are considered to occur at dose levels of less than 150 mg/kg bw.

As a result the substance is not considered to be classified for repeated oral toxicity under Directive 67/548/EEC, as amended for the 28th time in Directive 2001/59/EC. No need for classification for repeated-dose toxicity was seen when introducing the legal classification for this substance (Annex I index no. 607-281-00-4, ATP CLP00).

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008:

In the subacute oral toxicity study, effects were non-adverse at 50 mg/kg bw and adverse at 500 mg/kg bw. It is difficult to judge on the effects that would be seen at the cut-off dose level of 300 mg/kg bw. Considering the strong impact of peroxisome proliferation on the outcome of the study,

the substance is not considered to be classified for repeated oral toxicity under Regulation (EC) No. 1272/2008.

No need for classification for repeated-dose toxicity was seen when introducing the legal classification for this substance (Annex I index no. 607-281-00-4, ATP CLP00)

 

Dermal & Inhalation:

There are no data available.