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Toxicological information

Repeated dose toxicity: oral

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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:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
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

Reference
Name:
Unnamed
Type:
Constituent
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 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 / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
1 mg/kg bw/day
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
3 mg/kg bw/day
Basis:
nominal in diet
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.