Reaction mass of 2,2'-(vinylenedi-p-phenylene)bis[5-methylbenzoxazole] and 2,2'-(vinylenedi-p- phenylene)bisbenzoxazole and 2-[4-[2-[4-(benzoxazol-2- yl)phenyl]vinyl]phenyl]-5-methylbenzoxazole

Endpoint summary

Description of key information

Oral NOAEL (90d) (males and females): 1800 mg/kg bw/day

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

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:

From July 21 to November 04, 1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

Test item was given to SPF-Wistar rats in the daily diet over a period of 90 consecutive days; animals were distributed into 4 groups, each one composed by 20 males and 20 females. Nominal doses were 0, 200, 2000 and 20000 mg/kg diet; the individual feed mixtures were analyzed analytically for the content of test item. Behaviour, general state of health, body weight, food and drink consumption, heamatology analysis, clinical chemistry, urinalysis were assessed, as well as organs weights, gross and histopathology.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hoechst AG, Pharma-Forschung-Toxikoiogie, Kastengrund, gezüchtet unter SPF-Bedingungen.
- Age at study initiation: ca 4 - 6 weeks.
- Weight at study initiation: mean males 74.2 g (70 - 79 g); mean females 71.3 g (66 - 76 g).
- Housing: individually housed, in wire mesh cages (type 3), in a fully air-conditioned experimental room.
- Diet: Altromi n 1321-rodent diet, ad libitum.
- Water: drinking water, ad libitum.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 - 24 °C
- Humidity: 45 - 60 %
- Air changes: 6 times per hr.
- Photoperiod: 12 hours of artificial light (7.00 a.m. to 7.00 p.m.).

Route of administration:

oral: feed

Details on route of administration:

Nominal doses were 200, 2000 and 20000 mg/kg diet, which were prepared with 5000 mg test item with 24.995 kg of food Altromin 1321, 50000 mg test item with 24.950 kg of food Altromin 1321n and 500000 mg test item with 24.500 kg of food Altromin 1321, respectively.
At a distance of 7 days, the feed grains were emptied and refilled with dark-gelled feed.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The individual feed mixtures were analyzed analytically for the content of test item well as for homogeneity of the mixture.

Duration of treatment / exposure:

90 days

Dose / conc.:

200 mg/kg diet

Remarks:

males 17.27 mg/kg bw/day; females 18.30 mg/kg bw/day

Dose / conc.:

2 000 mg/kg diet

Remarks:

males 173.68 mg/kg bw/day; females 184.82 mg/kg bw/day

Dose / conc.:

20 000 mg/kg diet

Remarks:

males 1717.44 mg/kg bw/day; females 1864.27 mg/kg bw/day

No. of animals per sex per dose:

20 males and 20 females per group

Control animals:

yes, concurrent no treatment

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS
The behavior and the general state of health were assessed twice a day, on weekends and on holidays every day.
Weekly the rats were examined for possible disturbances of the tooth growth.

BODY WEIGHT
The body weight of all animals was determined at the beginning of the experiment and during the experiment once a week.

FOOD CONSUMPTION AND COMPOUND INTAKE
The feed consumption was determined continuously (1 weighing per week).
The measured values refer to the intervals between the body weight determinations. They are converted as a feed consumption per animal within 24 hours.

WATER CONSUMPTION
The water consumption was determined once a week. It is indicated as consumption per animal within 16 hours (15.15 - 7.15 clock).

OPHTHALMOSCOPIC EXAMINATION
Weekly the rats were examined for opacity of the eye media, damage to the oral mucosa.

HAEMATOLOGY
Heamatology analysis pre- and during the experiment was perfromed in 10 males and 10 females per group. The examinations after the end of the experiment and at the end of the observation period were carried out when animals were killed.
The blood was taken from the retroorbital venous plexus without any previous feed deprivation. The follow-up of cerium blood samples was randomized.
Parameters: hemoglobin, reticulocyte count, hematocrit, erythrocyte count, leukocytes, differential blood count, platelet count, clotting time and Heinz body.

CLINICAL CHEMISTRY
clinical chemistry analysis pre- and during the experiment was perfromed in 10 males and 10 females per group. The examinations after the end of the experiment and at the end of the observation period were carried out when animals were killed.
The values for Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH) and Mean corpuscular hemoglobin concentration (MCHC) were analyzed.

URINALYSIS
Urinalysis pre- and during the experiment was perfromed in 10 males and 10 females per group, as well as at the end of exposure period.
Parameters: appearance, colour, protein, glucose, hemoglobin, bilirubin, pH value, sediments and density.

NEUROBEHAVIOURAL EXAMINATION
Weekly the rats were examined for neurological disorders.

Sacrifice and pathology:

GROSS PATHOLOGY
Skin, body cavities, eyes, teeth, oral mucosa and internal organs were assessed macroscopically.
The absolute weight was recorded by the following organs and the relative weight was determined in relation to the body weight: heart, lung, liver, kidneys, spleen, brain, testes (without aids) / ovaries, adrenal, pituitary, thyroid and seminal vesicle.

HISTOPATHOLOGY
The following organs, or parts thereof, were introduced into fixation fluid and introduced into the histological examination: heart, lung, liver, kidneys, spleen, gland, jejunum, colon, urinary bladder, oesophageal, uterus, thyroid, pancreas, adrenal, thymus, hypophysis, brain, eye with opticus nerve, bone marrow.

Statistics:

The following measured values were statistically evaluated at a significance level of p = 0.05: body weights at the individual time points; body weight in the intervals day 9 - 29 and 36 - 92; haematological parameters, clinical chemistry parameters; relative organ weight.

Clinical signs:

no effects observed

Description (incidence and severity):

Throughout the experiment, behavior and general health were species-specific and not affected by the substance. Disturbances of the growth of the teeth, or changes in the oral mucous membrane due to the feeding of the substance were not recorded.
The excretion of the test substance occurred via the excrements, which were colored yellow as a function of the dose.

Mortality:

no mortality observed

Description (incidence):

Mortality did not occur during the experimental and follow-up period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

In all experimental animals, the development of the body weight was regular and during the whole experiment in good agreement with the body weight development of the control animals.

Description (incidence and severity):

The absolute and relative feed consumption showed no deviations from the standard in all treatment groups compared to the control.

Description (incidence and severity):

The relative consumption of drinking water was not influenced by test item intake.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Opacities of the eye-media was not recorded.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The following parameters showed deviations from the corresponding values of the control group: reduced erythrocyte count in both males and females, increased reticulocyte count in males, increased haemoglobin and reduced haematocrit in females only. However, a substance-related relevance can not be derived from the findings, since the changes are all within the narrow standard range and are only statistically significant.

The deviations in the male animals in the case of the erythrocytes (200 and 20000 mg/kg diet) and reticulocytes (2000 mg/kg diet) occurred independently of the dose; the slight increase in the number of reticulocytes in the male animals of the 2000 and 20000 ppm group at the end of the post-observation period, as well as the changes in the female animals should be considered in the context of the influence of age, at which erythrocytes, haematocrit and haemoglobin are continuously increased and the reticulocytes show correspondingly opposite behavior.
A comparison of the individual values clearly show that all the findings are within the narrow physiological standard range of the test animal strain.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

All the deviations shown in both male and female animals, with the exception of the glucose in the males, almost invariably occurred in a dose-free manner and all are within the narrow range of normality.
The glucose values in the male animals of the two highest doses (2000 and 20000 mg/kg diet) showed an increase of glucose up to the limit of the normal range; nevertheless the control values of the male and also the female rats to the end resulted to be in the same area.
Therefore, it can concluded that the clinical-chemical tests do not allow any evidence of substance-induced damage during the experiment.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

In some animals up to 1 mg haemoglobin/dl urine was found; very isolated, it was also found approximately 50 mg glucose/dl urine. These findings were not dose-related and occurred also in the control group.
A slight proteinuria (up to approx. 500 mg/dl) should be regarded as physiological in the rat strain used.
Bilirubinuria did not occur. The sediments were negligible. In animals with haemoglobin in the urine, erythrocytes, partly also leukocytes, were found. Epithelia occurred sporadically. The presence of bacteria is attributable to the collection technique (collection of urine over 16 hours).
In summary, it can be stated that no substance-induced changes in the urine status occurred.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Neurological disturbances were not recorded.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

In the postmortal studies, the relative brain weights of the female animals dosed at 200 mg/kg diet were significantly reduced; the relative lung weights of the males dosed at 200 mg/kg diet were significantly increased compared to the controls.
These changes were not found in higher dosage, thus they have been interpreted as independent to the substance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

The macroscopic and microscopic examination of the animals at the end of each experiment did not reveal any changes in the organism associated with the feeding of test item. In summary, it can be stated that the treatment did not lead to any morphologically detectable damage to the investigated organs in male and female rats, at none of the tested doses.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

The macroscopic and microscopic examination of the animals at the end of each experiment did not reveal any changes in the organism associated with the feeding of test item. In summary, it can be stated that the treatment did not lead to any morphologically detectable damage to the investigated organs in male and female rats, at none of the tested doses.

Dose descriptor:

NOEL

Effect level:

ca. 1 800 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

(20000 mg/kg diet)

Critical effects observed:

no

HAEMATOLOGY

Parameter	Dose (mg/kg diet)	Pre-treatment	Intermediate value	End of exposure	Recovery
Males
Erythrocyte count	200	n.s.	n.s.	-	n.s.
	20000	n.s.	n.s.	-	n.s.
Reticulocyte count	200	n.s.	n.s.	+	n.s.
	2000	n.s.	n.s.	n.s.	+
	20000	n.s.	n.s.	n.s.	+
Females
Erythrocyte count	200	-	n.s.	n.s.	n.s.
	20000	n.s.	n.s.	-	n.s.
Haemoglobin	2000	+	n.s.	n.s.	n.s.
Haematocrit	20000	n.s.	n.s.	-	n.s.

n.s.: not significant

+ significantly higher than the control group

- significantly lower than the control group

CLINICAL BIOCHEMISTRY

Parameter	Dose (mg/kg diet)	Pre-treatment	Intermediate value	End of exposure	Recovery
Males
Glucose	200	n.s.	-	+	n.s.
	20000	n.s.	n.s.	+	n.s.
SGOT	200	n.s.	+	n.s.	n.s.
	2000	-	+	n.s.	n.s.
Alkaline phosphatase	2000	-	n.s.	n.s.	-
Na	2000			n.s.	-
Ca	20000			n.s.	+
Cl	2000			n.s.	-
Inorg. Phosphate	200			n.s.	+
Total ilirubin	200			n.s.	-
	2000			n.s.	-
	20000			+	n.s.
Uric acid	200			+	n.s.
	20000			n.s.	+
Keratinine	2000			+	n.s.
	20000			+	n.s.
Females
Urea	200	n.s.	n.s.	-	n.s.
SGOT	200	n.s.		n.s.	n.s.
SGPT	200	n.s.	n.s.	-	n.s.
	2000	n.s.	n.s.	-	n.s.
	20000	n.s.	n.s.	-	-
Na	200			n.s.	+
K	200			n.s.	+
	20000			+	n.s.
Ca	200			+	n.s.
	2000			n.s.	-
Cl	20000			n.s.	+
Inorg. Phosphate	200			+	+
	2000			+	n.s.
Total ilirubin	2000			-	n.s.
	20000			-	n.s.
Keratinine	2000			-	-

n.s.: not significant

+ significantly higher than the control group

- significantly lower than the control group

Conclusions:

NOAEL (90d) (males and females): 1800 mg/kg bw/day

Executive summary:

In a study of systemic toxicity, test item was given to SPF-Wistar rats in the daily diet over a period of 90 consecutive days. Animals were distributed into 4 groups, each one composed by 20 males and 20 females. Nominal doses were 0, 200, 2000 and 20000 mg/kg diet; the individual feed mixtures were analyzed analytically for the content of test item well as for homogeneity of the mixture. Therefore, the actual doses were calculated to be 17.27 - 18.30 mg/kg bw/day, 173.68 - 184.82 mg/kg bw/day and 1717.44 - 1864.27 mg/kg bw/day for males and females, respectively.

Behavior and general health, body weight development and feed and drinking water consumption did not show any substance-induced changes in all experimental groups during the entire experimental period.

The determination of the organ weights and the macro- and microscopic examinations of the animals at the end of the experiment did not reveal any alteration in the organs.

Thus, the No Observed Effect Level was identified with the highest dosage 20000 mg/kg diet (i.e. ca 1800 mg/kg bw/day).

Conclusion

NOAEL (90d) (males and females): 1800 mg/kg bw/day

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 800 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

A sub-chronic test conducted with Fluorescent Brightener 219 is available; test item was given to SPF-Wistar rats in the daily diet, over a period of 90 consecutive days. Nominal doses were 0, 200, 2000 and 20000 mg/kg diet and the actual doses were calculated to be 17.27 - 18.30 mg/kg bw/day, 173.68 - 184.82 mg/kg bw/day and 1717.44 - 1864.27 mg/kg bw/day for males and females, respectively.

Behaviour and general health, body weight development and feed and drinking water consumption did not show any substance-induced changes in all experimental groups during the entire experimental period. The determination of the organ weights and the macro- and microscopic examinations of the animals at the end of the experiment did not reveal any alteration in the organs.

Thus, the No Observed Adverse Effect Level was identified with the highest dosage 20000 mg/kg diet (i.e. ca 1800 mg/kg bw/day).

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), 3.9 Specific target organ toxicity - repeated exposure section, substances are classified as specific target organ toxicants following repeated exposure by the use of expert judgment, on the basis of the weight of all evidence available, including the use of recommended guidance values, which take into account the duration of exposure and the dose/concentration, which produced the effect(s), and are placed in one of two categories, depending upon the nature and severity of the effect(s) observed.

In order to help reach a decision about whether a substance shall be classified or not, and to what degree it shall be classified (Category 1 or Category 2), dose/concentration ‘guidance values’ are provided for consideration of the dose/concentration which has been shown to produce significant health effects. The guidance values refer to effects seen in a standard 90-day toxicity study conducted in rats.

 

In the specific case, the No Observed Adverse Effect Level was established at 1800 mg/kg bw/day, on the basis of the results from the sub-chronic study on rat; therefore, the substance does not meet the criteria to be classified for repeated dose toxicity, according to the CLP Regulation (EC 1272/2008).