Dinoseb

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2 May 1977 to 2 January 1979.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1981

Report date:

1981

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

The only major deviation from the OECD 408 guideline was that the animals were subject to two mating periods at the end of the 13 week (91 day) dosing period, and were not subject to necropsy until week 29 (Ca. day 203).

Principles of method if other than guideline:

The study was conducted as a 3 generation reproductive and developmental toxicity study with a 13 week (91 day) dosing period, followed by two mating periods for a total dosing period of 29 weeks. The substance was administered orally in feed at concentrations to provide 1, 3 or 10mg/kg bw/day nominally. The only major deviation from the OECD 408 guideline was that the animals were subject to two mating periods at the end of the 13 week (91 day) dosing period, and were not subject to necropsy until week 29 (Ca. day 203). This deviation is considered to reduce the reliability of the result for use in this regulatory endpoint, however the result is still considered to be adequately reliable.

GLP compliance:

no

Remarks:

Study pre-dates GLP however, Quality Assurance audits/inspections were performed during the study and included as a QA Audit record within the report

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Dinoseb

EC Number:

201-861-7

EC Name:

Dinoseb

Cas Number:

88-85-7

Molecular formula:

C10H12N2O5

IUPAC Name:

dinoseb

Details on test material:

The test article, a brown crystalline solid (batch number MM 200025), was supplied in a Winchester by the study sponsor. The test article was melted by allowing hot water to run over the container. When the contents had melted, the Winchester was agitated to ensure homogeneity and the test article was decanted into a number of wide-necked containers, which were stored in a domestic refrigerator when not in use. The study was carried out using this one sample of test article.
Purity was determined to be 98%

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Sprague Dawley derived rats of the CD strain obtained from a specific pathogen-free colony were used for the study. They were obtained from Charles River (UK) Ltd., Manston Road, Margate, Kent.
- Age at study initiation: (P) = ~40 days old at the start of the study
- Weight at study initiation: (P) Mean bw = Males: 191 g, Females: 158 g; (F1) Males: 86 g; Females: 80 g; (F2) Males: 67 g; Females: 64 g
- Housing: The animals were housed in solid floor polypropylene cages with stainless steel lids (North Kent Plastics, Dartford, Kent: dimensions 56 cm x 38 cm x 18 cm). Autoclaved soft wood chips were provided for bedding (supplied by the Sawdust Marketing Company, Standon, Herts.). Before parturition, mated females were provided with shredded tissue paper for nesting material.
Number of animals per cage:
Males - Females
pre-mating periods 5 - 5
mating periods (1 x 1) paired together
gestation periods 5 - 1
lactation periods 5 - 1 + litter
behavioural animals 6 - 6

- Diet (e.g. ad libitum): The basic diet used was Rat and Mouse Diet No. 1 Expanded and Reground (B.P. Nutrition, Stepfield, Witham, Essex) obtained in powdered form and offered in non-spill hoppers. After commencement of the study, the manufacturer modified the diet to increase the vitamin K level from 1ppm to 10ppm. This modified diet was used from the 35th week of the study until termination.
- Water (e.g. ad libitum): The animals had free access to mains water available from glass bottles attached to each cage. The water was changed daily and the bottles were replaced at weekly intervals with clean sterilised bottles.
- Acclimation period: The animals were acclimatised to the laboratories for a period of 4 days prior to the start of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 50 ± 10%
- Air changes (per hr): approximately 18 air changes per hour
- Photoperiod (hrs dark / hrs light): The animals were exposed to an artificial fluorescent photoperiod of 12 hours light : 12 hours dark.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Batches of test diets were prepared at weekly intervals, although this schedule was altered infrequently to accommodate holidays or other unforeseen factors and unused test diet was discarded at the end of each week.
With the exception of the mating periods when male and female animals were housed together, separate diets were prepared for each dose level and sex, and a separate aliquot of test article was weighed for each dosage level.
- Mixing appropriate amounts with (Type of food): The required amount of test article was ground in a mortar with a small amount of powdered diet to form a mixture of high test article concentration. This concentrate was made up to the final weight with the remaining powdered diet and mixed in either a Morton '50E' batch mixer or a Gardner 3C double cone blender for approximately 15 minutes.
- Storage temperature of food: The formulated test diets were stored in galvanised metal bins with tightly fitting lids. This storage condition was necessary in view of the known property of the test article to migrate into plastic.
The stability of the test article in the powdered diet was carried out by HLE.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

No analysis of diet formulations were made in this study. Stability of Dinoseb in the diet when stored at room temperature for 7 days was demonstrated.

Duration of treatment / exposure:

The test diets were continuously available over 3 generations (87 weeks), the F0 generation was dosed for approximately 13 weeks prior to mating followed by dosing over the 2 mating periods lasting another 16 weeks for a total of 29 weeks (Ca. 203 days) dosing period.

Frequency of treatment:

ad libitum in diet

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
1 mg/kg bw/day
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
3 mg/kg bw/day
Basis:
nominal in diet

Doses / concentrations 3

Remarks:

Doses / Concentrations:
10 mg/kg bw/day
Basis:
nominal in diet

No. of animals per sex per dose:

50 (25 male/25 female)

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The levels of the test material were selected by the sponsor, based upon worst-case environmental and food exposures.

Positive control:

Not applicable.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
All animals were examined at least once daily to determine their general health and behaviour. Any changes observed were recorded on an individual clinical record.
Any animal that appeared to be ill was isolated to permit further observation. If its condition improved, the animal was returned to the group cage; if the death of an animal appeared probable, it was killed. These animals together with any found dead (with the exception of pre-weaned pups) were subjected to a macroscopic necropsy.


DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: The body weights of non-pregnant, weaned animals were recorded at weekly intervals throughout the study (although this schedule was altered infrequently to accommodate holidays or other unforeseen factors).
The body weights of pregnant animals were recorded on days 0, 3, 6, 9, 12, 15, 18 and 21 of gestation. The body weights of lactating animals were recorded on days 1, 7, 14 and 21 of lactation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
The group food consumption of the selected F0, F1 and F2 generation animals was recorded weekly throughout the study (although this schedule was altered infrequently to accommodate holidays or other unforeseen factors).
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Other examinations:

none.

Statistics:

Adult bodyweight was analysed for co-variance.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

- Bodyweights - males:
Treatment with Dinoseb at 10 mg/kg/day (high dose) over 3 generations elicited a marked retardation of body weight gain in comparison with controls. The difference in body weight between the control and high dose males was approximately 20% in the F0 phase.
High dose males showed a marked retardation in the rate of weight gain in comparison with the controls and other treated groups from week 3. By week 13, high dose males were significantly lighter than the controls and, at the end of the F0 phase, the high dose males were approximately 20% lighter than the controls.
In comparison with the control group, intermediate dose males showed a similar rate of body weight gain, however low dose animals showed a slightly lower weight gain.

- Body weights - females:
Treatment with Dinoseb at 10 mg/kg/day (high dose), elicited a retardation of body weight gain in comparison with controls.
The effect was less marked in each of the succeeding generations.
High dose females showed a marked retardation in the rate of weight gain in comparison with the controls and other treated groups from week 3. By week 14, the high dose females were significantly lighter than the controls. The retardation was evident throughout mating, gestation and lactation of both the first (F1a) and second (F1b) litters. Similar rates of weight gain to the control group were observed in the low and intermediate dose groups.

- Food consumption:
There was no evidence of treatment related intergroup differences in group mean food intake for the males during the periods reported.
For the first 13 weeks of the F0 generation there was no evidence of treatment related intergroup differences in group mean food intake for the females.

- Clinical observations/mortalities - males:
All the intercurrent deaths were considered incidental and not related to treatment with Dinoseb. Clinical changes occurred in the surviving animals in all dose groups, including the controls, and were mainly lesions caused by fighting or nasal and ocular discharges. Males in the high dose group in all generations generally developed a yellow tinge to the coat.

- Clinical observations/mortalities - females:
All the intercurrent deaths were considered incidental and not related to treatment with Dinoseb. Clinical changes in the surviving animals were mainly minor in nature and occurred in all dose groups including the controls. Females in the high dose group in all generations generally developed a yellow tinge to the coat.

- Macroscopic findings at necropsy, microscopic findings and organ weights:
In the adult males, the observed lesions were primarily associated with the pulmonary tissue and the incidence did not suggest an effect of treatment with Dinoseb at any of the dose levels employed.
Observed lesions in the adult females were generally of pulmonary origin and the intergroup distribution did not suggest an effect of treatment with Dinoseb at any of the dose levels employed.
The only treatment related observation at necropsy was in the group treated at 10 mg/kg/day Dinoseb. The majority of the animals in this group developed a yellow tinge to the fur. This change appeared to have resulted from direct contact of the fur with the test article (which was yellow-brown in colour) in the diet.

- Mating performance and fertility:
There was no effect of treatment with Dinoseb on male or female fertility. Fertility of both sexes was similar to the controls in all 3 generations.
Fecundity in the Dinoseb treated groups was similar to the controls in each mating phase in all 3 generations.
There was little intergroup variation in mating performance in either mating phase in each generation.

F0 generation:
In the first mating (F0-F1a), the fecundity indices were 96.0% in the low dose group and 100% in the control, intermediate and high dose groups. Mating indices were 100%, 96.2%, 92.3% and 100% in the control, low, intermediate and high dose groups, respectively.
In the second mating (F0-F1b) the fecundity indices were 100%, 88%, 100% and 96% in the control, low, intermediate and high dose groups, respectively. Mating indices were 100% in each group.
The male fertility index for the F0 generation was 100% in each group and the female fertility index was 96% in the control group and 100% in each of the low, intermediate and high dose groups.

Effect levels

Dose descriptor:

NOAEL

Effect level:

3 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Bodyweight gain in F0 generation and a yellowish tinge to the fur in all generations.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1. Calculated test article intake (mg/kg/day).

Week number	Group 1 Control 0 mg/kg/day Dinoseb	Group 2 1 mg/kg/day Dinoseb	Group 3 3 mg/kg/day Dinoseb	Group 4 10 mg/kg/day Dinoseb
1	0	1.2	3.96	12.9
2	0	1.27	3.42	12.34
3	0	0.85	3.06	8.73
4	0	1.08	2.96	11.32
5	0	0.95	3.03	10.47
6	0	1.2	3.32	11.36
7	0	0.91	2.99	9.66
8	0	0.94	2.98	9.57
9	0	0.93	3.12	8.54
10	0	*	*	*
11	0	0.93	3.52	10.02
12	0	1.01	3.35	11.02
13	0	1.06	3.45	10.13
14-65	0	*	*	*
66	0	1.07	3.09	10.07
67	0	0.98	2.64	10.55
68	0	1.05	3.51	9.54
69	0	0.97	3.03	10.34
70	0	0.99	2.88	9.56
71	0	1	3.05	10.2
72x	0	0.55	1.8	6.85
73x	0	0.63	1.54	5.49
74	0	0.91	3.19	11.71
75	0	0.88	2.42	7.82
76	0	1.21	4.06	13.19
77	0	1	2.89	11.16
78	0	0.89	4.11	9.19
79	0	1.24	2.07	8.28
80x	0	0.74	1.77	6.64
81x	0	0.51	1.65	5.3
82	0	0.67	2.31	8.79
83	0	1.11	2.58	9.33
84	0	1.17	3.7	12.3
85	0	0.96	2.75	9.92
86	0	0.93	3.05	10.63
87	0	0.89	2.52	8.32

* - Records not available.

x - mating period: males eat female diet during this period and are consequently under dosed.

Table 2. Calculated test article intake (mg/kg/day).

Week number	Group 1 Control 0 mg/kg/day Dinoseb	Group 2 1 mg/kg/day Dinoseb	Group 3 3 mg/kg/day Dinoseb	Group 4 10 mg/kg/day Dinoseb
1	0	1.67	5.02	17.84
2	0	1.16	3.56	11.8
3	0	0.96	2.78	10.19
4	0	0.93	3	9.09
5	0	1.29	4.13	12.65
6	0	1.24	3.59	11.67
7	0	1.12	3.28	10.17
8	0	1.01	3.4	11.93
9	0	1.11	3.07	9.65
10	0	*	*	*
11	0	0.8	3.07	8.84
12	0	0.87	3.38	10.36
13	0	1.8	4.26	13.66
14-65	0	*	*	*
66	0	1.07	3.07	10.14
67	0	1	3.51	11.91
68	0	1.08	2.42	8.61
69	0	0.91	2.98	10.34
70	0	0.96	3.03	9.15
71	0	1.12	2.91	10.4
72x	0	0.86	2.94	10.54
73x	0	1.01	2.58	8.69
74	0	1.24	4.34	11.68
75L	0	0.73	2.3	7.06
76L	0	1.58	4.54	11.67
77L	0	1.16	3.61	15.4
78L	0	1.79	4.92	15.96
79L	0	1.58	5.09	16.65
80x	0	1.17	3.07	10.4
81x	0	0.85	2.99	8.69
82	0	0.73	2.75	9.04
83L	0	1.72	4.67	13.97
84L	0	1.92	4.94	13.76
85L	0	1.44	3.72	12.58
86L	0	1.4	3.71	13.17
87L	0	1.57	3.99	12.6

x - mating period.

L - littering/lactation: food intake artificially elevated as pups wasting and eating food.

* - record not available.

Volume 2: Table 1. Group mean bodyweights (g) F₀ males.

Period (weeks)	Group 1 Control 0 mg/kg/day Dinoseb	Group 2 1 mg/kg/day Dinoseb	Group 3 3 mg/kg/day Dinoseb	Group 4 10 mg/kg/day Dinoseb
1	191	190	193	191
2	239	249	236	229
3	291	300	295	281
4	327	336	343	310
5	258	356	367	329
6	281	365	387	351
7	418	412	419	372
8	444	430	441	387
9	460	455	469	401
10	473	461	476	403
11	484	477	479	400
12	507	493	504	415
13	524	501	518	427
14	516	498	506	409
15	529	511	526	440
16	519	503	519	440
17	530	510	527	443
18	535	517	532	443
19	547	534	536	468
20	534	524	530	453
21	541	537	536	442
22	555	552	558	455
23	576	560	573	488
24	591	569	591	507
25	597	571	585	501
26	598	574	590	503
27	598	574	590	505
28	601	579	596	509
29	624	595	616	503

Volume 2: Table 2. Group mean bodyweights (g) F₀ females (F₀ → F₁a)

Period		Group 1 Control 0 mg/kg/day Dinoseb	Group 2 1 mg/kg/day Dinoseb	Group 3 3 mg/kg/day Dinoseb	Group 4 10 mg/kg/day Dinoseb
Pre-mating (weeks)	1	160	157	158	155
	2	185	186	186	178
	3	211	220	220	212
	4	226	218	219	210
	5	237	228	233	221
	6	249	239	243	229
	7	266	256	259	241
	8	269	261	260	240
	9	283	273	275	257
	10	281	271	273	255
	11	287	279	276	255
	12	292	282	282	264
	13	302	288	289	268
	14	304	289	290	266
Gestation period (days)	0	297	291	291	272
	3	308	304	301	281
	6	317	309	309	287
	9	327	320	318	297
	12	324	326	325	298
	15	342	341	337	315
	18	378	377	370	346
	21	405	405	398	373
% bodyweight increase over gestation period		36.4	39.2	36.8	37.1
Lactation period (days)	1	306	308	301	280
	7	326	329	321	305
	14	309	317	313	299
	21	294	307	299	282
Gestation period (days)	0	310	313	309	290
	3	325	329	322	303
	6	333	336	331	309
	9	344	344	342	316
	12	360	360	355	329
	15	373	378	370	343
	18	411	418	409	381
	21	446	450	440	410
% bodyweight increase over gestation period		43.9	43.8	42.4	41.4
Lactation period (days)	1	341	344	347	314
	7	346	363	356	331
	14	358	372	360	344
	21	344	354	350	334

Applicant's summary and conclusion

Conclusions:

No effects attributed to administration of Dinoseb were observed in the evaluation of parental survival, necropsy findings, fertility, fecundity or in the various reproductive indices examined. The NOAEL value of 3 mg/kg bw/day (the middle tested dose) was based upon a statistically significant reduction in the bodyweight gain of animals in the highest dose group (10 mg/kg bw/day) together with the appearance of a yellowish tinge in the fur of animals of the same group.

Executive summary:

A 3 generation reproduction studywas carried out in the Sprague-Dawley derived rat.

Groups of 25 male and 25 female rats in each of the 3 generations (F0, F1 and F2) received Dinoseb in the diet at concentrations to provide 1, 3 or 10 mg/kg bw daily for 29 weeks (total duration 87 weeks). Similar groups of animals-fed untreated diet over the same period served as the control group.

The parents of all 3 generations were fed the appropriate diets for 13 weeks and then subjected to 2 mating trials. The offspring from the first mating of each parental generation (F1a, F2a and F3a) were maintained until weaning and then killed and necropsied. A proportion of the parental females from each group and generation were killed on day 21 after the second mating for teratological observation.

Criteria evaluated for test article effect included parental body weight, survival, clinical signs, pregnancy incidences, reproductive indices, caesarean data and necropsy finding

Selected offspring were also evaluated for post-natal functional development. No effects attributed to administration of Dinoseb were observed in the evaluation of parental survival, necropsy findings, fertility, fecundity or in the various reproductive indices examined. The NOAEL value of 3 mg/kg bw/day (the middle tested dose) was based upon a statistically significant reduction in the bodyweight gain of animals in the highest dose group (10 mg/kg bw/day) together with the appearance of a yellowish tinge in the fur of animals of the same group.