Fluometuron

Endpoint summary

Description of key information

Subchronic (90-day) study oral (gavage), rat (CDF/CrlBR (F-344)) m/f, (OECD guideline 408 (1981)): NOAEL: = 7.5 ppm equivalent to mean daily intakes of 5.9 mg/kg/day (male) and 6.5 mg/kg/day  (female)
Subacute (28-day) study dermal (occlusive), rabbit (New Zealand White) m/f, ((EU Method B.9 (Repeated Dose (28 Days) Toxicity (Dermal)): NOEL: 1000  mg/kg bw/day (male/female)

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:

22 November 2001 to 14 June 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline study The experiments were done according to the Organisation for Economic Co-operation and Development, testing of Chemicals Guideline No. 408 (revised 1998) EU 2001/59/EEC, Method B.26

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Species:

rat

Strain:

Fischer 344/DuCrj

Sex:

male/female

Details on test animals or test system and environmental conditions:

-group of 55 male and 55 female rats of the Fisher CDF/CrlBR (F-344) strain
- Source: Harlan UK Limited (UK), Bicester, Oxfordshire, England
- each animal was assigned a number and a tail tattoo
- weight: males: 108 - 135 g, females: 96 to 117 g
- age: 26 - 30 days
- free access to mains drinking water and food
- animal room temperature: 19 - 23°C
- relative humidity: 40 - 70%

Route of administration:

oral: feed

Vehicle:

other: diet

Details on oral exposure:

The test material was administered via the diet continuously throughout the treatment period.
Animals did not have access to mixed diet beyond the end of its shelf-life, as determined by the stability test. Control animals received untreated diet at the same frequency, and from the same batch of basal diet, as treated animals. During the recovery period, all animals were given untreated basal diet.
A record of the weight of each formulation dispensed and the amount remaining after feeding was made. The balance was compared with the predicted usage as a check that the diet had been administered correctly. No significant discrepancy was found.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Fluometuron was prepared for administration as a series of graded concentrations in powered basal diet and was incorporated to provide the required concentrations by initial preparation of a premix at a concentration of 15000 ppm. The amount of Fluometuron required for the premix was added to an equal amount of sieved diet and stirred. An amount of sieved diet equal to the weight of the mixture was added and the mixture was stirred again until visibly homogenous; the doubling up process was repeated until approximately half the premix diet was added. At wich stage the mixture was ground with a coffee grinder. The mixture was made up to the weight of the premix with coarse diet and was then mixed using a turbula T10 mixer.
The premix was diluted with the further quantities of coarse diet to prepare the two highest concentrations (750 ppm and 7500 ppm) and a second premix was used to prepare the lowest concentration test mix (75 ppm).
Batches of the diets were prepared weekly and issued in sealed metal containers. The unused residucs were discarded at the end of each treatment weeek.
Detailed records of compound usage were maintened. The amount of test material necessary to prepare the formulations and the amount actually used were determined on each occasion of preparation. The difference between these amounts was checked before the formulations were dispensed.
Before treatment commenced, the suitability of the proposed mixxing procedure was determined and specimen formulations were analysed to assess the homogeneity and stability of the test material in the diet matrix. Samples of each formulation prepared for administration Weeks 1, 6 and 12 of the treatment period were analysed for achieved concentration of the test substance. The method of analysis was an adaption of a method supplied by the Sponsor.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

continuously throughout the treatment period

Remarks:

Doses / Concentrations:
0, 75, 750 and 7500 ppm (equivalent to 0, 5.9, 60.9, 586, and 0, 6.5, 66.7, 617 mg/kg/day for males and females respectively) for 13-weeks
Basis:
nominal in diet

No. of animals per sex per dose:

A group of twenty rats (ten males and ten females) was dosed as follows: 75 ppm, Main study (13 weeks)
A group of twenty rats (ten males and ten females) was dosed as follows: 750 ppm, Main study (13 weeks)
A group of twenty rats (ten males and ten females) was dosed as follows: 7500 ppm, Main study (13 weeks)
A group of twenty rats (ten males and ten females) was dosed as follows: Control: 0 ppm, Main study (13 weeks)
A group of ten rats (five males and five females) was dosed as follows: 7500 ppm, Recovery phase (4 weeks)
A group of ten rats (five males and five females) was dosed as follows: Conrol: 0 ppm, Recovery phase (4 weeks)

Control animals:

yes, concurrent no treatment

Observations and examinations performed and frequency:

Four groups of ten male and ten female rats to CDF/CrlBR (F-344) rats received fluometuron in the diet at concentrations of 0, 75, 750 and 7500 ppm (equivalent to 0, 5.9, 60.9, 586, and 0, 6.5, 66.7, 617 mg/kg/day for males and females respectively) for 13-weeks. Two additional groups of, five male and 5 female rats received fluometuron in the diet at concentrations of 0 and 7500 ppm for 13-weeks followed by a four-week recovery period without fluometuron.
The homogeneity and stability of the test substance in the diet was assessed before the study commenced. Samples of each formulation prepared for administration in Weeks 1, 6 and 12 were analysed for achieved concentration of the test substance.
During the study, clinical observation, bodyweight, food consumption, detailed physical and arena observations, sensory reactivity and grip strength, motor activity, ophthalmic examination, haematology, blood chemistry, urinalysis, organ weight, macroscopic and microscopic pathology investigations were undertaken.
Findings see tables 7.5.1-02 to 7.5.1.-05

Sacrifice and pathology:

Organ weight, macroscopic and microscopic pathology investigations were undertaken

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no mortalities during the treatment or recovery periods. Clinical signs related to treatment were restricted to hairloss on the head and dorsal body surface up to approximately Week 6 in males receiving 7500 ppm. Yellow staining in cages housing animals receiving 750 or 7500 ppm was seen throughout the treatment period, with occasional yellow staining also observed in the early part of the treatment period in cages housing animals receiving 75 ppm.

Mortality:

mortality observed, treatment-related

Description (incidence):

There were no mortalities during the treatment or recovery periods. Clinical signs related to treatment were restricted to hairloss on the head and dorsal body surface up to approximately Week 6 in males receiving 7500 ppm. Yellow staining in cages housing animals receiving 750 or 7500 ppm was seen throughout the treatment period, with occasional yellow staining also observed in the early part of the treatment period in cages housing animals receiving 75 ppm.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Initial bodyweight loss was apparent amongst animals receiving 7500 ppm diet; The bodyweights of animals receiving 75 or 750 ppm were considered unaffected by treatment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Throughout the treatment period, lower than control food intake was evident amongst animals receiving 7500 ppm diet. The bodyweights of animals receiving 75 or 750 ppm were considered unaffected by treatment.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Overall food conversion efficiency was low for males and females that received 7500 ppm. There was no effect of treatment upon food conversion efficiency in animals receiving 75 or 750 ppm.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There was no ophthalmic finding during Week 12 of treatment that was considered to have arisen as a result of treatment with fluometuron.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Haemoglobin concentration, erythrocyte counts and mean cell haemoglobin concentration were reduced for animals receiving 750 or 7500 ppm (week 13)

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

A dose-related increase in the total plasma cholesterol concentration in treated males and in females receiving 7500 ppm. This change was not evident at the end of the recovery period.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Urinary volume was decreased for males and females receiving 7500 ppm. Urinary protein concentrations were decreased for males receiving 750 or 7500 ppm, but were slightly increased for females receiving 7500 ppm.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

After 13 weeks of treatment, the absolute and bodyweight-relative spleen weights of females given 75 ppm and of males and females given 750 or 7500 ppm were high when compared with the controls.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

dark kidneys in animals given 7500 ppm and swollen, irregular, dark or enlarged spleen amongst animals that had received 7500 ppm; dark spleens were apparent in animals treated at 750 ppm.

Details on results:

MORTALITIES
There were no mortalities during the treatment or recovery periods

CLINICAL SIGNS
Clinical signs related to treatment were restricted to hairloss on the head and dorsal body surface up to approximately Week 6 in males receiving 7500 ppm. Yellow staining in cages housing animals receiving 750 or 7500 ppm was seen throughout the treatment period, with occasional yellow staining also observed in the early part of the treatment period in cages housing animals receiving 75 ppm.

MOTOR ACTIVITY
Motor activity assessment in Week 12 indicated that the number of high beam breaks (rearing) in male animals receiving 7500 ppm was lower than the controls. This change was still evident at the end of the 4 week recovery period, at this time these animals additionally had lower numbers of low beam breaks (cage floor activity levels). Sensory reactivity and grip strength were unaffected by treatment.

BODY WEIGHT
Initial bodyweight loss was apparent amongst animals receiving 7500 ppm diet; subsequently, poor bodyweight gain was evident in these animals. At the end of the 13-week treatment period the weight gain of these animals was reduced by approximately 45%, compared with the controls. During the recovery period, previously treated animals showed significantly higher bodyweight gain than the control animals, however, the bodyweights for these animals were still lower than those of the controls indicating that full recovery had not occurred during the 4-week recovery period.
The bodyweights of animals receiving 75 or 750 ppm were considered unaffected by treatment. (See Table 7.5.1-01: Summary of body weight – group mean values (g))

FOOD CONSUMPTION
Throughout the treatment period, lower than control food intake was evident amongst animals receiving 7500 ppm diet. The largest variation from control occurred during the first week of treatment. During the recovery period, food consumption of animals that had previously received 7500 ppm diet was essentially similar to that of the controls.
The bodyweights of animals receiving 75 or 750 ppm were considered unaffected by treatment.

FOOD CONVERSION EFFCIENCY
Overall food conversion efficiency was low for males and females that received 7500 ppm; Week 1 food conversion efficiency could not be calculated due to weight loss during this week. During the recovery period, higher than Control food utilisation was apparent amongst animals that had previously received 7500 ppm.
There was no effect of treatment upon food conversion efficiency in animals receiving 75 or 750 ppm

HAEMATOLOGY
During Week 13 of treatment when compared with controls, haemoglobin concentration, erythrocyte counts and mean cell haemoglobin concentration were reduced for animals receiving 750 or 7500 ppm; the mean cell volume of these animals was increased. In females receiving 750 or 7500 ppm there was a reduction in the haematocrit. Mean cell haemoglobin was high, compared with the controls, in females receiving 750 ppm and in males and females receiving 7500 ppm. When compared with controls, eosniophil counts were low and monocyte counts were high for animals receiving 750 or 7500 ppm, lymphocyte counts were high for females receiving 750 or 7500 ppm, platelet counts were increased for females receiving 750 ppm and reduced for males and females receiving 7500 ppm. In animals receiving 7500 ppm there was an increase in prothombin time and an increase in activated partial thromoplastin time amongst females receiving 7500 ppm. A number of changes in the blood film after 13 weeks of treatment were apparent. The incidence of anisocytosis, macrocytosis, hypochromasia, Howell-Jolly Bodies and microcytic cell fragments was increased in animals receiving 7500 ppm; normoblasts were evident in one male and three females. Greater than control anisocytosis incidence was apparent amongst males receiving 750 ppm; animals receiving 75 ppm were unaffected. After four weeks of recovery, high haematocrit, haemoglobin concentration, erythrocyte counts and mean cell haemoglobin concentrations that were similar to controls indicated that full recovery had occurred. Mean cell haemoglobin and mean cell volume remained slightly high though there was evidence of partial recovery since the difference from controls was less than that observed at the end of the treatment period. None of the treatment-related changes in blood morphology was evident at the end of the treatment period.
Platelet counts remained lower than controls at the end of the recovery period in both sexes previously given 7500 ppm. The magnitude of this change was similar to that observed at the end of the treatment period, indicating no significant recovery had occurred. The variations in prothrombin and activated partial thromboplastin times and lymphocyte and monocyte counts previously evident amongst females treated with 7500 ppm were not evident indicating that full recovery had occurred.

BLOOD CHEMiSTRY
Blood chemistry examination during Week 13 of treatment revealed, when compared with controls, a dose-related increase in the total plasma cholesterol concentration in treated males and in females receiving 7500 ppm. This change was not evident at the end of the recovery period.
Plasma creatinine concentrations were decreased in treated males and in females receiving 7500 ppm. The inter-group difference from controls for males receiving 75 or 750 ppm was, however, small and unlikely to be of any toxicological significance. At the end of the recovery period, creatinine concentrations were still low in previously treated males, though the difference was less than that observed at the end of the treatment period, indicating partial recovery; females showed full recovery.
In females receiving 7500 ppm there was a slight increase, compared with the controls, of plasma glucose. This change was fully reversible.
Plasma potassium concentrations were increased in treated males and in females receiving 7500 ppm. The inter-group difference from controls for males receiving 75 or 750 ppm was very small and of no toxicological significance. The change in plasma potassium showed full recovery.
In both sexes there was a marginal increase in albumin concentration at 7500 ppm and a concomitant increase in the albumin to globulin ratio. These differences were not evident at the end of the recovery period. A number of other inter-group differences attained statistical significance, compared with the controls, but they were minor or lacked dosage-relationship and were therefore considered to represent normal biological variation. Such changes included the minimally low plasma alanine amino-transferase activities and sodium concentrations in males receiving 7500 ppm and the variations of plasma urea in females. (See Table 7.5.1-02: Summary of significant blood film comments after 13 weeks of treatment)

URINE ANALYSIS
During Week 13 of treatment, when compared with controls, urinary volume was decreased for males and females receiving 7500 ppm. Urinary protein concentrations were decreased for males receiving 750 or 7500 ppm, but were slightly increased for females receiving 7500 ppm. The difference in females is not considered biologically significant. This effect was not present at the end of the recovery period.
An effect upon the appearance of the urine was evident at 750 and 7500 ppm. The urine of animals receiving 750 or 7500 ppm appeared darker than that of the controls at the end of the treatment period. The degree of darkening was dose-related. By the end of the recovery period there was evidence of some recovery as the degree of darkening in animals previously given 7500 ppm was less that that observed at the end of the recovery period.
When compared with the controls, urinary potassium concentrations were decreased for animals receiving 7500 ppm; this was not evident at the end of the recovery period.
Other changes in the urine at the end of the treatment period, such as the slightly increased pH in treated females, were considered to reflect normal variation. (See Table 7.5.1.-03: Summary of urinary appearance)

ORGAN WEIGHTS
After 13 weeks of treatment, the absolute and bodyweight-relative spleen weights of females given 75 ppm and of males and females given 750 or 7500 ppm were high when compared with the controls. At the highest dietary concentration of fluometuron the effect upon spleen weight was particularly marked. The bodyweight-relative kidney weights of females given 75 or 750 ppm and of males and females given 7500 ppm were higher than those of the controls. In addition, at 7500 ppm, bodyweight-relative liver weights were high in both sexes, absolute and bodyweight-relative adrenal weights were low in females and absolute and bodyweight-relative thymus weights were low in males.
A number of changes in organ weight at 7500 ppm were attributed to the large inter-group difference in bodyweight. Such changes included the variations of brain, epididymides, heart, testes, ovary and uterus weights.
After four weeks of recovery, the spleen weights of animals previously given 7500 ppm were still higher than those of the controls, though the difference was less than that seen at the end of the treatment period. All other differences in organ weight that were attributed to treatment were no longer apparent at the end of the recovery period.

MACROPATHOLOGY
Macroscopic examination after 13 weeks of treatment revealed a high incidence, when compared with controls, of dark kidneys in animals given 7500 ppm and swollen, irregular, dark or enlarged spleen amongst animals that had received 7500 ppm; dark spleens were apparent in animals treated at 750 ppm. Thin uterus, small ovaries and thickened splenic capsules were evident amongst females receiving 7500 ppm. After 4 weeks of recovery, the high incidence of swollen and irregular spleen amongst animals that had previously given 7500 ppm was still evident as was thin uterii amongst females that had previously received 7500 ppm. Other treatment-related macroscopic abnormalities evident after 13 weeks of treatment had shown recovery. (See Table 7.5.1.-04: Summary of treatment-related macroscopic findings)

MICROPATHOLOGY
Treatment-related microscopic findings were restricted to the kidney, liver, spleen, ovaries and uterus of animals receiving 750 or 7500 ppm.
Renal cortical tubular pigment and pigment in the Kupffer cells were apparent in animals receiving 7500 ppm and also in females treated at 750 ppm, although the severity was reduced. Investigation with special stains (Perls Prussian Blue and Schmorl’s) revealed the presence of two pigments, haemosiderin and lipofuscin. These findings were still evident after four week recovery period at similar incidences, however, the severities were reduced.
Splenic congestion, capsular inflammatory cell infiltrate and thickening were apparent following treatment with either 750 or 7500 ppm. These findings were still evident after the four week recovery period at similar incidences, however, the severities were reduced.
Sparse or absent corpora lutea and uterine endometrial atrophy were apparent in females treated at 7500 ppm; the endometrial atrophy was also evident in one female receiving 750 ppm. The atrophy was still evident after the four-week recovery period at a similar incidence, however, the ovarian findings were no longer apparent.

Dose descriptor:

NOAEL

Effect level:

ca. 5.9 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Dose descriptor:

NOAEL

Effect level:

ca. 6.5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Critical effects observed:

not specified

Table 7.5.1 -02: Summary of body weight – group mean values (g)

Sex	Male				Female
Dose level (ppm)	0	75	750	7500	0	75	750	7500
Achieved intake (mg/kg/day)	0	5.9	60.9	586.0	0	6.5	66.7	617.3
Week
0	120	120	122	120	106	106	107	107
1	143	145	146	111	119	120	120	100
2	172	172	173	130	134	136	134	114
3	193	195	195	143	143	144	141	118
4	213	215	213	153	150	151	148	121
5	233	236	234	166	160	162	158	130
6	246	250	247	175	164	167	161	133
7	262	266	261	185	170	173	168	137
8	275	279	272	194	175	176	171	143
9	285	284	279	200	176	176	172	140
10	295	294	290	212	181	182	178	147
11	302	299	296	216	182	183	180	147
12	311	307	303	225	185	187	183	151
13	308	303	300	221	184	186	183	148
Bodyweight gain
0-1	23	25	24	-9*	13	14	13	-7*
1-13	165	159	154	110*	65	65	64	49*
0-13	188	183	178	101*	77	80	77	42*
Bodyweight gain as % of control
	-	97	95	54	-	104	100	55

Significant when compared to control * p < 0.01

Table 7.5.1 -03: Summary of significant blood film comments after 13 weeks of treatment

Sex	Male				Female
Dose level (ppm)	0	75	750	7500	0	75	750	7500
Anisocytosis	1	1	3	6	0	0	0	2
Macrocytosis	0	0	0	5	0	0	0	8
Hypochromasia	0	0	0	10	0	0	0	15
Howell-Jolly bodies	0	0	0	4	0	0	0	1
Microcytic cell fragments	0	0	0	14	0	0	0	15
Normoblasts	0	0	0	1	0	0	0	3
Number of animals examined	15	10	10	15	15	10	10	15

Table 7.5.1.-04: Summary of urinary appearance

	Main study (13 weeks)				Recovery		Main study (13 weeks)				Recovery
Sex	Male						Female
Dose level (ppm)	0	75	750	7500	0	7500	0	75	750	7500	0	7500
Normal colourless/pale yellow	13	9	5	1	0	0	14	5	2	0	0	0
Cloudy pale yellow	2	1	2	0	5	0	1	5	7	0	0	0
Medium yellow/cloudy medium yellow	0	0	3	9	0	5	0	0	1	6	0	4
Dark yellow/ cloudy dark yellow	0	0	0	5	0	0	0	0	0	9	0	0
Number of animals examined	15	10	10	15	5	5	15	10	10	15	5	5

Table 7.5.1.-05: Summary of treatment-related macroscopic findings

	Main study (13 weeks)				Recovery		Main study (13 weeks)				Recovery
Sex	Male						Female
Dose level (ppm)	0	75	750	7500	0	7500	0	75	750	7500	0	7500
Kidneys
- Dark	0	0	0	8	0	0	0	0	0	7	0	0
Spleen	0	0	0	1	0	5	0	0	1	0	0	4
- Swollen	0	1	1	10	0	3	0	0	4	10	0	4
- Irregular surface	0	0	0	10	0	5	0	0	0	10	0	5
- Dark	0	0	4	10	0	0	0	0	5	10	0	0
- Enlarged	0	0	0	4	0	1	0	0	0	9	0	0
- Capsule thickened	0	0	0	0	0	1	0	0	0	5	0	0
Ovaries
- Small
Uterus
- Thin	-	-	-	-	-	-	1	0	1	10	0	2
Number examined	10	10	10	10	5	5	10	10	10	10	5	5

Conclusions:

It is concluded that dietary administration to F-344 rats of fluometuron for 13 weeks was well tolerated up to 750 ppm, but caused a marked toxic response at 7500 ppm. The principal target of toxicity was the erythrocyte. Findings at 75 ppm were minor and limited to yellow staining of the cage trays, attributed to the presence of a coloured urinary metabolite, and slightly increased liver and spleen weights, though there was no associated histopathological change. Consequently, 7.5 ppm is considered to represent the no-observed-adverse-effect level (NOAEL) in this study (equivalent to 5.9 mg/kg/day and 6.5 mg/kg/day in males and females respectively). With the exception of reduced platelet counts, all findings related to treatment showed full recovery, or evidence that recovery was underway, during the recovery period.

Executive summary:

The systemic toxic potential of the test substance, Fluometuron (a systemic herbicide against broadleaf and grass weeds of cotton), to CDF®/CrlBR (F-344) rats by dietary administration was assessed over a period of 13-weeks. The recovery from any treatment-related effect was assessed during a subsequent four-week recovery period. Three groups of ten male and ten female rats received Fluometuron in the diet at concentrations of 75, 750 or 7500 ppm for 13-weeks. A similarly constituted Control group received the basal diet alone. A further five males and five females were assigned to each of the Control group and the group receiving 7500 ppm; these animals were treated for 13-weeks, followed by a four-week period without treatment.

 

During the study, clinical condition, bodyweight, food consumption, detailed physical and arena observations, sensory reactivity and grip strength, motor activity, ophthalmic examination, haematology, blood chemistry, urinalysis, organ weight, macroscopic and microscopic pathology investigations were undertaken.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

5.9 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

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 April to 16 May 1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

No study guidelines are reported. The study has the following deficiencies when compared with the toxicity study in rats, oral route (method B.31 of Regulation (EC) No 440/2008): The study duration was 21 days compared to the required 28 days, the number of tissues taken for histopathology was less extensive than required, there is no reference to dose rate selection and the area treated is not recorded.

Qualifier:

no guideline followed

Guideline:

EU Method B.9 (Repeated Dose (28 Days) Toxicity (Dermal))

Deviations:

yes

Remarks:

The study duration was 21 days compared to the required 28 days, the number of tissues taken for histopathology was less extensive than required, there is no reference to dose rate selection and the area treated is not recorded

Principles of method if other than guideline:

The study duration was 21 days compared to the required 28 days, the number of tissues taken for histopathology was less extensive than required, there is no reference to dose rate selection and the area treated is not recorded

GLP compliance:

yes

Remarks:

self certification by laboratory

Limit test:

yes

Species:

rabbit

Strain:

New Zealand White

Sex:

male/female

Details on test animals or test system and environmental conditions:

- group of five male and five female New Zealand white rabbits (SPF)
- Source: Hazleton Research Animals, Denver, P.A.
- weight: 2.42 - 3.38 kg
- age:twelve to sixteen weeks old
- free access to mains drinking water and food
- animal room temperature:67 +/- 5°F
- relative humidity: 30 - 70%

Type of coverage:

occlusive

Vehicle:

water

Details on exposure:

Groups of five female and five male New Zealand White albino rabbits received the test material as a 6-hour topical application to the clipped skin under occlusive bandages at doses of 0, 10, 100 or 1000 mg/kg for 21 days. The test material was applied as a paste in water.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

6-hour topical application/day for 21 days

Frequency of treatment:

procedures were completed daily from day 1 to the day prior sacrifice

Remarks:

Doses / Concentrations:
0, 10, 100, 1000 mg/kg
Basis:
nominal per unit body weight

No. of animals per sex per dose:

VEHICLE CONTROL: Dose level 0 mg/kg: 5 male and 5 female/dose
Treatment I: Dose level 10 mg/kg: 5 male and 5 female/dose
Treatment II: Dose level 100 mg/kg: 5 male and 5 female/dose
Treatment III: Dose level 1000 mg/kg: 5 male and 5 female/dose

Control animals:

yes, concurrent no treatment

Details on study design:

Groups of five female and five male New Zealand White albino rabbits received the test material as a 6-hour topical application to the clipped skin under occlusive bandages at doses of 0, 10, 100 or 1000 mg/kg for 21 days. The test material was applied as a paste in water:

Animals were randomly assigned to study groups prior to study initiation using a computer program employinq a method adapted from Carnaham, Luther, and Wilkes, Applied Numerical Methods, Wiley, 1969. Only animals considered to be in good health and with application sites free of abnormalities were included in the randomization procedure. Because of exclusion due to poor skin at the application site an an1mal with an eye abnormality was used on this study. To prevent confounding the results of the ophthalmology studies this animal was manually assigned to the vehicle control group.

Animal Preparation and Dosing Procedures
The back of each animal was clipped free of hair using electric clippers equipped with a number 40 (surgical) blade prior to initial applications of the test article paste. The application sites were reclipped twice each week during the study to ensure good material to skin contact.
As each test and vehicle control group animal was manually restrained, the pre-weighed test article and water paste was applied to the clipped application area and spread as evenly as possible over the test site using a wooden spatula. Any paste remaining in the individual test article container and on the wooden spatula was wiped off with a gauze pad. This gauze pad was then placed over the application site. The gauze pad and test article were held in place using a cellophane binder. This binder encircled the abdomen and was secured with several overlapping bands of masking tape at the anterior and posterior ends of the binder. Vehicle control animals were treated similarly except no test article paste was applied. Prior to applyinq binders to the vehicle control animals, the application site was moistened with the vehicle (distilled deionized water). After 6 hours of exposure, the adhesive tape, plastic binder, and gauze were removed and the application site was gently wiped with a gauze pad soaked in water to remove any remaining test article. These procedures were completed daily from day 1 to the day prior to sacrifice with the following exceptions:
- A low dose female was underdosed by approximately 13% for 5 consecutive days due to a calculation error.
- Wrapping materials were inadvertently not removed until the morning following application from 1 mid dose male on day 6.
Neither of the listed deviations from standard application procedure were considered to have an adverse impact on the outcome of the study.

Clinical Indices
All animals were observed for mortality and overt signs of toxicity twice daily during the study in the early morning and late afternoon. Any abnormalities observed during the twice daily checks or observed at any other time during the study were recorded. Prior to each day's treatment, the application site on each animal was evaluated for erythema and edema according to Draize. Any other dermal reactions were also recorded. Individual body weights were measured weekly. Animals found dead were weighed prior to necropsy. Terminal fasted body weights were recorded on the days of scheduled sacrifice.

Clinical pathology Studies
Blood samples (obtained via the orbital sinus) were collected from all animals prior to study initiation and prior to scheduled sacrifice. All animals were fasted for approximately 16 hours prior to blood collection.
Animals were randomly assigned to study groups prior to study initiation using a computer program employinq a method adapted from Carnaham, Luther, and Wilkes, Applied Numerical Methods, Wiley, 1969. Only animals considered to be in good health and with application sites free of abnormalities were included in the randomization procedure. Because of exclusion due to poor skin at the application site an an1mal with an eye abnormality was used on this study. To prevent confounding the results of the ophthalmology studies this animal was manually assigned to the vehicle control group.

Animal Preparation and Dosing Procedures
The back of each animal was clipped free of hair using electric clippers equipped with a number 40 (surgical) blade prior to initial applications of the test article paste. The application sites were reclipped twice each week during the study to ensure good material to skin contact.
As each test and vehicle control group animal was manually restrained, the pre-weighed test article and water paste was applied to the clipped application area and spread as evenly as possible over the test site using a wooden spatula. Any paste remaining in the individual test article container and on the wooden spatula was wiped off with a gauze pad. This gauze pad was then placed over the application site. The gauze pad and test article were held in place using a cellophane binder. This binder encircled the abdomen and was secured with several overlapping bands of masking tape at the anterior and posterior ends of the binder. Vehicle control animals were treated similarly except no test article paste was applied. Prior to applyinq binders to the vehicle control animals, the application site was moistened with the vehicle (distilled deionized water). After 6 hours of exposure, the adhesive tape, plastic binder, and gauze were removed and the application site was gently wiped with a gauze pad soaked in water to remove any remaining test article. These procedures were completed daily from day 1 to the day prior to sacrifice with the following exceptions:
- A low dose female was underdosed by approximately 13% for 5 consecutive days due to a calculation error.
- Wrapping materials were inadvertently not removed until the morning following application from 1 mid dose male on day 6.
Neither of the listed deviations from standard application procedure were considered to have an adverse impact on the outcome of the study.

Clinical Indices
All animals were observed for mortality and overt signs of toxicity twice daily during the study in the early morning and late afternoon. Any abnormalities observed during the twice daily checks or observed at any other time during the study were recorded. Prior to each day's treatment, the application site on each animal was evaluated for erythema and edema according to Draize. Any other dermal reactions were also recorded. Individual body weights were measured weekly. Animals found dead were weighed prior to necropsy. Terminal fasted body weights were recorded on the days of scheduled sacrifice.

Clinical pathology Studies
Blood samples (obtained via the orbital sinus) were collected from all animals prior to study initiation and prior to scheduled sacrifice.
All animals were fasted for approximately 16 hours prior to blood collection.

Observations and examinations performed and frequency:

All animals were observed for mortality and overt signs of toxicity twice daily during the study in the early morning and late afternoon.
Blood samples (obtained via the orbital sinus) were collected from all animals prior to study initiation and prior to scheduled sacrifice.

Sacrifice and pathology:

ORGAN WEIGHTS
The following organs brain, adrenal, heart, kidney, liver, pituitary on both males and female animals were weighed at sacrifice and the organ weights, organ to body weight ratio, organ to brain weight ratio were calculated. Additional determinations were made on the testes with epididymides in males and ovaries in females. The fasted final body weight was also recorded. No statistically significant differences were detected
OPHTHLOMOLOGY
A small posterior capsular cataract was observed in a female dosed at 100 mg/kg. This was recorded as an incidental finding.
GROSS NECROPSY
There were no gross necropsy findings in the tissues of any group, which could be attributed to treatment with fluometuron technical. All findings were considered to be either spontaneous or consistent with a population of age matched experimental rabbits.
Two animals, one female found dead at Day 15 treated at 100 mg/kg and one male treated at 100 mg/kg at sacrifice showed evidence of enteritis. Both cultured positive for Klebsiella pneumonia. The findings did not adversely affect the evaluation of dermal toxicity of fluometuron.
HISTOPATHOLOGY
The histopathology observations made in both control and treated animals included inflammatory lesions of the kidney, tubular degeneration in the testes. There was also, low grade responsive and inflammatory changes of the skin which were of lower incidence when compared to the control. All of the findings appeared unrelated by incidence and concentration of test substance.
CLINICAL PATHOLOGY
No treatment related changes in haematology or clinical chemistry were detected from blood samples taken prior to sacrifice.

Statistics:

Body weight, organ weight, organ weight ratio, hematology and clinical chemistry data were statistically analyzed for group differences by either analysis or variance or Kruskal-Wallis Test (heterogenous or non-parametric data). Any significant differences were further analyzed using either Tukey´s (equal populations), Scheffe´s (unequal populations) or Kruskal-Wallis Test of Multiple Comparisons. Only significant pairwise comparisons between the control and treated groups were tabulated and discussed.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no treatment-related effects: One female at a dose level of 100 mg/kg was found dead on Day 15. The death was, believed to be attributed to acute bacterial enteritis and thus unrelated to treatment.

Dermal irritation:

no effects observed

Description (incidence and severity):

No treatment related skin reactions or incidence of erythema or oedema

Mortality:

no mortality observed

Description (incidence):

There were no treatment-related effects: One female at a dose level of 100 mg/kg was found dead on Day 15. The death was, believed to be attributed to acute bacterial enteritis and thus unrelated to treatment.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was also no difference in overall bodyweight gain between the groups

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was also no difference in food consumption between the groups

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related effects

Haematological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related effects

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no treatment-related effects

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no treatment-related effects

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Critical effects observed:

not specified

Table 7.5.3-01: Overview of 21 day repeated dose dermal toxicity in albino rabbits treated with fluometuron technical

Sex	Male				Female
Dose (mg/kg)	0	10	100	1000	0	10	100	1000
Mortality	There were no treatment-related effects*
Bodyweight*	There were no treatment-related effects*
Mean bodyweight (kg) Initial	2.21	2.20	2.19	2.18	2.17	2.19	2.16	2.14
Week 1	2.31	2.35	2.29	2.30	2.29	2.36	2.28	2.25
Week 2	2.42	2.45	2.43	2.41	2.46	2.56	2.37	2.44
Week 3	2.58	2.60	2.55	2.57	2.65	2.73	2.59	2.61
Mean total weight gain (kg)	0.37	0.39	0.36	0.38	0.48	0.54	0.44	0.47
Standard deviation	0.169	0.091	0.148	0.103	0.118	0.099	0.100	0.100
Mean final body weights (kg)	2.4	2.5	2.5	2.4	2.6	2.7	2.5	2.5
Standard deviation	0.21	0.18	0.21	0.13	0.18	0.33	0.19	0.25
Food consumption*
Mean (g/animal/day) Week 1	163.20	173.42	178.27	169.13	173.60	181.44	155.50	152.80
Week 2	169.60	171.53	165.87	172.33	183.67	193.67	131.89	168.67
Week 3	185.33	182.33	154.87	170.60	202.60	201.80	183.33	184.27
Ophthalmology	There were no treatment-related effects
Dermal reactions	No treatment related skin reactions or incidence of erythema or oedema
Clinical pathology (haematology)	There were no treatment related effects
Clinical pathology (clinical chemistry)*	There were no treatment related effects
Organ weights	There were no treatment related effects
Gross necropsy	There were no treatment related effects

*One female in 100 mg/kg treatment group was found dead on Day 15

Conclusions:

Fluometuron technical does not cause dermal irritation or systemic toxicity in the rabbit when administered for 21 days at doses up to 1000 mg/kg.
Under the conditions of this study the no observed effect level (NOEL) is proposed to be 1000 mg/kg/day

Executive summary:

Fluometuron Technical was evaluated for dermal irritation and systemic toxicity by topical application to the skin of albino rabbits under occlusive bandages for 6 hours/day on twenty-one consecutive days at doses of 10, 100, or 1,000 mg/kg. A concurrent vehicle control group was also evaluated.

One mid-dose female died on study day 15. This death was attributed to an acute bacterial enteritis judged to be unrelated to treatment.

Growth and food intake were unaffected by treatment. No skin reactions were observed that could be attributed to treatment and there were no treatment related changes in hematology or clinical chemistry parameters. No ophthalmoscopic effects were observed.

No significant differences were observed between control and test group organ weight or organ weight ratio data. There were no treatment related gross or microscopic findings.

In conclusion, Fluometuron Technical, when administered at dermal doses of up to 1000 mg/kg, did not produce any dermal irritation or systemic toxicity in the rabbit.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Additional information

Subchronic (90-day) study oral (gavage), rat (CDF/CrlBR (F-344)) m/f, (OECD guideline 408 (1981)):

The rat was chosen as the best species because of its acceptance as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The CDF/CrlBR (F-344) strain was used because of a historical control data available in the laboratory. The 90-day study with a 28-day recovery period administration of fluometuron, in the diet to CDF/Crl BR (F-344) rats for 13-weeks was well tolerated up to 750 ppm, but produced marked toxicity at 7500 ppm. The main target of toxicity was the erythrocyte.

Overall the principal short term effects of fluometuron were body weight loss or reduced gain in the highest dose levels tested due to reduced food consumption. Target organs were the liver, spleen, kidneys. The principal target of the toxicity of fluometuron is considered to be the erythrocyte. The broadly similar effects seen as enlarged liver, spleen and kidneys, with dark discoloration and the associated congestion and deposition off haemosiderin can be attributed to an increase in the rate of erythrocyte turnover. In a 90-day rat study (Rees, 2003) there is evidence that fluometuron affected erythrocyte synthesis, resulting in instability of the erythrocyte membrane, since most high dosage animals (7500 ppm, rat ) showed microcytic fragmentation in the peripheral blood. It is possible that this is a result of mechanical damage to the erythrocyte, as an adverse effect upon the membrane would reduce membrane deformability, thereby limiting the ability of the erythrocyte to pass though smaller blood vessels and the spleen. Further evidence of some impairment of erythrocyte formation is the presence of Howell-Jolly Bodies in both the rat and dog. The deposition of haemosiderin and lipofuscin in the liver and kidney is considered to be due to storage of the iron component of haemoglobin and an increase in the turnover of subcellular organelles as seen in actively metabolising cells possibly as a consequence of haemoglobin metabolism. There is evidence of brown pigment in hepatic Kupffers cells in all high dose animals and renal tubular epithelium in high dose females.

Fluometuron technical does not cause dermal irritation or systemic toxicity in the rabbit when administered for 21 days at doses up to 1000 mg/kg. Under the conditions of the study the no observed effect level (NOEL) is proposed to be 1000 mg/kg/day

Justification for classification or non-classification

No mortalities were observed within repeated dose studies (dermal/oral) and all animals showed full recovery within observation period. Inhalation study was not performed as concentration range was technically limited within acute inhalation study to 4.62 mg/L without any mortality. Nevertheless route-to-route extrapolation was chosen to calculate DNEL.

In respect to regulation 1272/2008, Annex I, 3.1.2 Fluometuron is not classified as being toxic.