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Description of key information

- 28 d repeated dose oral toxicity: NOAEL ≥ 300 mg/kg bw/day, OECD TG 407 28 d, gavage

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1999-08-20 to 1999-03-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: - OECD TG 407 compliant - GLP compliant
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
as at 27 July 1995
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
as at 30 September 1996
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Strain: Rats, Fischer, CDF(F344)/CRLBR, SPF
- Source: Charles River Wiga GmbH, D-97633 Sulzfeld, Germany
- Age at study initiation: ca. 6 weeks
- Weight at study initiation: Males: 131.1 ± 8.9 g; Females: 106.0 ± 8.8 g
- Fasting period before study: no
- Housing: single caging in Makrolon cages type III (39 cm x 23 cm x 18 cm); wire mesh lids, sanitation of the cages once a week; aspen wood chips, type "4 HV" (Finn Tapvei Oy, SF-73620 1 Kortteinen), autoclaved; bedding material changed weekly
- Diet (e.g. ad libitum): ad libitum, Altromin 1314 forte, gamma irradiated with 25 kGy 60Co, Altromin GmbH, D-32791 Lage
- Water (e.g. ad libitum): ad libitum, tap water, acidified with HC1 to pH 3, from an automatic watering system
- Acclimation period: 13 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 44
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- made freshly each day shortly before the administration to the animals by dissolution of test substance in deionised water

VEHICLE
- Justification for use and choice of vehicle (if other than water): water
- Concentration in vehicle: 3, 9.5, and 30 mg/mL
- Amount of vehicle (if gavage): 10mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
HPLC-UV/Vis
- solutions of the test substance derivated for 2.5 hours at 110 °C (derivating reagent: 160 mg NaOH, 2.5 ml acetyl acetone, dissolved in 1000 ml methanol)
- System:
• Hewlett Packard 1050 system,
• Integrator HP3395
• Column: YMC-Pack ODS-A, 5 gm (150 x 6 mm), produced by YMC Co., Kyoto, Japan
• Temperature: ambient
• Flow: 1.0 mL/min
• Mobile phase: 10.2 g KH2PO4 , 250 mL acetonitrile and 2.5 mL triethylamine, ad 750 mL deionised water
• Injection volume: 20 µL
• Retention time: ca. 5 min
• Detection: UV detector set at 295 nm
Duration of treatment / exposure:
28 d, recovery groups kept further 16 d without treatment
Frequency of treatment:
7 d/wk
Remarks:
Doses / Concentrations:
30, 95 and 300 mg/kg bw per day
Basis:
actual ingested
No. of animals per sex per dose:
10/sex at control and 300 mg/kg bw/day group, 5/sex at 30 and 95 mg/kg bw/day groups; 5/sex of control and high dose group were kept as recovery groups
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on pre-experiments; no toxicity signs in a 7 d dose range finding study at 50, 158 and 500 mg, but all animals died within 2 d at 1000 mg/kg without specific toxic effects or indications of target organ toxicity.
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: high dose group and controls
- Post-exposure recovery period in satellite groups: 16 d
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations:
• general signs
• health status
• viability (twice daily)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: d -1, 6, 13, 20, 27: all animals of all groups, d 34, 41: all animals of the recovery groups
- Detailed clinical observations checked in table 2 were included.

BODY WEIGHT: Yes
- Time schedule for examinations: d -6, d 1, 8, 15, 22, 28 (all groups) and on 29, 35 and 43 (recovery groups only)

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: d -6, d 1-7, 7-21, 21-28 (all groups) and d 29- 35 and 35-43 (recovery groups only)

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: d 29 (all dose groups except recovery groups)
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes, over night
- How many animals: all animals of all dose groups except recovery groups
- Parameters checked in table 3 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: d 29 (all dose groups except recovery groups)
- Animals fasted: Yes, over night
- How many animals: all animals of all dose groups except recovery groups
- Parameters checked in table 3 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: d 27
- Dose groups that were examined: all dose groups
- Battery of functions tested:
• behaviour, of the motor activities and sensory reactivity to different stimuli (acoustic, tactile, visual and proprioceptive) tested outside the home cage in a standard arena.
• eyelid and auricular reflexes (tactile and proprioceptive)
• acoustic reaction (clapping of hands)
• visual reactivity
• righting reflex
• forelimb grip strength

OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 4), conducted on d 29 all dose groups except recovery groups and on d 44 for recovery groups
HISTOPATHOLOGY: Yes (see table 4), conducted on d 29 all dose groups except recovery groups and on d 44 for recovery groups
Other examinations:
no
Statistics:
- Tests:
• Analysis of variance followed by the Scheffe test: all data with means and standard deviations determined, for comparison of more than two groups
• t-test: all data with means and standard deviations determined, for comparison of two groups only
• H-test of Kruskal and Wallis followed by the test of Nemenyi: counted events with scoring or in cases where the requirements for the analysis of variance were not fulfilled.
• Chi²-test: counted events
• Fisher's exact test: counted events, if the Chi2-Test was not applicable
- Results analysed separately for males and females; P = 0.05 chosen in each test; two tailed test were used. Groups K and KR and C and CR were treated separately for statistical analysis.
- Numerical data have been rounded for presentation, a manual recalculation therefore may yield slightly different results to those given in the tables.
Details on results:
CLINICAL SIGNS AND MORTALITY
- small lesions (term "dermal wounds or crusts", single lesions with a size of less than 1 mm) on the skin of the tails noted in life in several dosed animals, significant differences in the incidence of these alterations present only in high dosed animals of both sexes compared to the controls
- alterations attributed to biting or scratching by the animals themselves, probably caused by local itching.
- only some indications of reduced well-being (raised fur) noted in high dosed animal
- incidental findings (chromodakryorrhoea, respiratory murmur) not attributed to the action of the test substance
- see table 7 for details

BODY WEIGHT AND WEIGHT GAIN
- body weights of the high dosed males significantly reduced from Day 15 onwards until the end of the recovery period
- no significant group differences nor a corresponding trend noted in body weights of high dose females
- see table 8 for details

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
- significantly less food consumption in the first 2-3 weeks of the study in high dose males
- no significant group differences nor a corresponding trend noted in body weights of high dose females

HAEMATOLOGY
- no significant or dose related group differences in haematology

CLINICAL CHEMISTRY
- no significant or dose related group differences in clinical chemistry.

NEUROBEHAVIOUR
- no significant differences between dosed groups and the controls or dose related trends noted at functional observations or in grip strength

ORGAN WEIGHTS
statistically significant differences relative to controls
- high dose males:
• decrease: in absolute brain weight after recovery, absolute heart weight after 28 d, heart weight relative to brain weight after 28 d,
• increase: in brain weight relative to bw, testes relative to bw after 28 d and after recovery, kidneys relative to bw after 28,
- high dose females:
• decrease: -
• increase: thymus absolute weight and relative to bw after recovery
- see table 9 for details

GROSS PATHOLOGY
- only known spontaneous changes without any dose relationship noted at necropsy

HISTOPATHOLOGY: NON-NEOPLASTIC
- no significant or dose related group differences noted histopathologically.
- skin of the tails, changed in life: histopathologically normal; indication for secondary irritation to scratching or nibbling rather than for primary dermal lesion
Dose descriptor:
NOAEL
Effect level:
>= 300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects no substance related adverse effects at highest dose.
Critical effects observed:
not specified

- Table 4: Survey of the daily observations in life.

finding

Number of affected males/females in group

 mg/kg bw

0

(d 27)

0 recovery (d 41)

30

(d 27)

95

(d 27)

300

(d 27)

300 recovery (d 41)

dermal wounds, crusts a

0/0

0/0

0/1

1/0

5/5

0/0

Relevant observations are presented by number of affected animals.

(a) multiple, tiny erosions on the tails.

- Table 5: Mean body weights

Data are presented by mean, standard deviation (sd) and number of animals (n).

mg/kg bw

para-meter

body weight (g) on Days 22

-6

1

8

15

22

28

35

43

Males

0

mean

131.2

167.2

193.6

213.6

232.8

242.8

-

-

 

sd

7.9

6.9

6.1

6.3

7.3

9.4

 

 

 

n

5

5

5

5

5

5

 

 

0 recovery

mean

131.2

164.8

192.8

214.6

232.4

243.0

253.6

263.8

 

sd

7.3

8.0

8.2

9.1

10.8

11.0

9.9

11.4

 

n

5

5

5

5

5

5

5

5

30

mean

131.6

162.8

186.8

206.6

225.2

234.4

-

-

 

sd

9.9

9.9

8.9

8.9

9.0

9.5

 

 

 

n

5

5

5

5

5

5

 

 

95

mean

129.0

160.8

184.8

204.0

223.0

230.2

-

-

 

sd

10.0

13.8

14.2

13.5

14.9

15.8

 

 

 

n

5

5

5

5

5

5

 

 

300

mean

132.8

166.2

181.4

190.8*

207.4*

209.8*

-

-

 

sd

9.2

8.0

12.0

10.8

8.3

8.8

 

 

 

n

5

5

5

5

5

5

 

 

300 recovery

mean

130.6

164.2

175.8

191.6*

208.4*

216.6*

226.6*

239.6*

 

sd

8.2

14.1

14.9

8.5

11.0

12.3

13.0

10.8

 

n

5

5

5

5

5

5

5

5

Females

0

mean

107.2

123.4

133.0

140.8

149.0

151.0

-

-

 

sd

7.9

6.9

6.1

6.3

7.3

9.4

 

 

 

n

5

5

5

5

5

5

 

 

0 recovery

mean

106.0

124.6

134.0

143.0

149.6

152.8

159.0

160.2

 

sd

7.3

8.0

8.2

9.1

10.8

11.0

9.9

11.4

 

n

5

5

5

5

5

5

5

5

30

mean

105.1

121.2

130.8

139.0

146.0

150.6

-

-

 

sd

9.9

9.9

8.9

8.9

9.0

9.5

 

 

 

n

5

5

5

5

5

5

 

 

95

mean

106.2

123.2

134.0

143.4

150.4

154.8

-

-

 

sd

10.0

13.8

14.2

13.5

14.9

15.8

 

 

 

n

5

5

5

5

5

5

 

 

300

mean

105.3

121.4

131.2

136.8

143.6

147.0

-

-

 

sd

9.2

8.0

12.0

10.8

8.3

8.8

 

 

 

n

5

5

5

5

5

5

 

 

300 recovery

mean

106.3

124.6

134.0

139.4

148.0

152.2

159.2

161.2

 

sd

8.2

14.1

14.9

8.5

11.0

12.3

13.0

10.8

 

n

5

5

5

5

5

5

5

5

* Significant differences to the control

for Table 6 see under "Overall remarks, attachments" (this is only necessary due to space limitations in this field)

Conclusions:
Body weights and feed consumption were reduced in high dosed males compared to the control. However, the effects were reversible in the recovery group and body weight gain reduction was less than 10% at the end of the recovery period compared to control recovery males. Organ weight/body weight ratio was increased in brain, testis and kidneys of high dose group males, which was attributed to the reduced body weight gain. Histopathological alterations of the organs were not found and all organ weight effects showed high tendency of reversibility in the recovery group. In the majority of the males and female in high dose group dermal wounds were found on tails which cleared up completely during recovery period. Due to the high grade of effect reversibility in the recovery group no adverse effects (LOAEL) of the test substance could be determined.
Based on the results of this study, the "No observed adverse effect level" (NOAEL) is higher than or equal to 300 mg Guanidine carbonate per kg body weight and day in both sexes.
Executive summary:

Guanidine carbonate was tested in a 28 d repeated dose study in rats with application via the oral route according to OECD Guideline 407 and GLP. The test substance was administered as a solution in deionised water orally by gavage to 3 groups of 5 male and 5 female F344 rats each, once a day on 7 days per week for 28 consecutive days.

Doses used:

•         Group A (low dose): 30 mg per kg body weight and day,

•         Group B (mid dose): 95 mg per kg body weight and day,

•         Group C (high dose): 300 mg per kg body weight and day.

An equally sized negative control group (group K) received deionised water, the vehicle for the test substance. The dose volume was uniformly 10 ml per kg body weight. In addition, two groups of 5 males and 5 females each, i.e. one high dose recovery group (group CR) and one control recovery group (group KR), were treated in the same way as their corresponding groups, but were kept then for further 14 days without test substance administration in an attempt to observe the reversibility, persistence or delayed onset of test substance induced lesions.

Investigations:

•         Animal observations: once a day (plus a daily check for viability).

•         Detailed clinical observations: once a week.

•         Functional observations: once in the last week of the dosing period.

•         Body weight: once a week.

•         Feed consumption: for each week.

•         Haematology: on Day 29 and on Day 44.

•         Clinical biochemistry: on Day 29 and on Day 44.

•         Necropsy with gross pathological examination: on Day 29 and on Day 44.

•         Organ weight determination.

•         Histopathological examination.

Results

•         Mortality:

All animals survived until the scheduled termination.

•         Animal observation, detailed clinical examination, functional tests:

Dermal alterations (tiny wounds or crusts) were noted occasionally on tails of animals in all dosed groups, but mainly in high dosed group males and females. This effect cleared up completely at the end of the recovery period. Except for this, only unspecific signs were noted.

•         Body weights:

In high dosed males a significantly lower body weight was present from Day 15 on. The effect was reversible in the recovery group and body weight gain reduction was less than 10% at the end of the recovery period compared to control recovery males

No significant differences or dose related trends were noted in the body weights of the females.

•         Feed consumption:

In high dosed males a significantly lower feed consumption was present from the start of dosing but recovered completely in the recovery group. No significant differences or dose related trends were noted in the feed consumption of the females.

•         Haematology and clinical chemistry:

No significant differences were noted between any dosed group and the controls.

•        Functional observations:

No significant differences were noted between any dosed group and the controls.

•         Necropsy with gross pathological examination and histopathology:

No treatment related alterations were noted. The altered skin areas of the tails were normal histopathologically.

  •       Organ weight determination:

Organ weight/body weight ratio was increased in brain, testis and kidneys of high dose group males, which was attributed to the reduced body weight gain. The effects showed high tendency of reversibility in the recovery animals.

Conclusion

Based on the results of this study, the "No observed adverse effect level" (NOAEL) is higher than or equal to 300 mg Guanidine carbonate per kg body weight and day in both sexes.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
- OECD TG 407 compliant
- GLP compliant

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

Oral repeated dose toxicity

Guanidine carbonate was tested in a 28 d repeated dose study in rats with application via the oral route (gavage) at doses of 30, 95 and 300 mg/kg bw/d conducted according to OECD TG 407 and GLP.

Body weights and feed consumption were reduced in high dosed males compared to the control. However, the effects were reversible in the recovery group and body weight gain reduction was less than 10 % at the end of the recovery period compared to control recovery males. Organ weight/body weight ratio was increased in brain, testis and kidneys of high dose group males, which was attributed to the reduced body weight gain. Histopathological alterations of the organs were not found and all organ weight effects showed high tendency of reversibility in the recovery group. In the majority of the males and female in high dose group dermal wounds were found on tails which cleared up completely during recovery period. Due to the high grade of effect reversibility in the recovery group no adverse effects (LOAEL) of the test substance could be determined.

Based on the results of this study, the "No observed adverse effect level" (NOAEL) is ≥ 300 mg Guanidine carbonate per kg body weight and day in both sexes.

Inhalation repeated dose toxicity

No inhalation repeated dose toxicity studies are available.

Dermal repeated dose toxicity

No dermal repeated dose toxicity studies are available.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
A GLP study performed in accordance with OECD TG 407 is available and was chosen as the key study.

Justification for classification or non-classification

Based on the results of the study the substance does not need to be classified according to CLP (Regulation (EC) No 1272/2008).