Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May- December 1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP study, certificate of analysis of poor quality, no biochemical and histological observations.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report date:
1989

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
: Histopathology, haematology, biochemistry and sensory reactivity analyses were not carried out.
Principles of method if other than guideline:
Not applicable.
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetrakis(hydroxymethyl)phosphonium sulphate(2:1)
EC Number:
259-709-0
EC Name:
Tetrakis(hydroxymethyl)phosphonium sulphate(2:1)
Cas Number:
55566-30-8
Molecular formula:
C4H12O4P.1/2O4S
IUPAC Name:
tetrakis(hydroxymethyl)phosphonium sulphate(2:1)
Test material form:
other: liquid stored at room temperature in the dark
Details on test material:
- Name of test material (as cited in study report): the test substance is DP 435 Biocide. DP 435 is the development code for the active substance Tolcide PS75.
- Physical state: clear colourless liquid
- Stability under test conditions: yes
- Storage condition of test material: stored at room temperature

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)BR strain (VAF plus)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, UK
- Age at study initiation: not reported
- Weight at study initiation: 116 to 154 g for females, 117 to 165 g for males
- Fasting period before study: no data
- Housing: by groups of 5 animals, per sex, in grid bottomed stainless steel cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 25°C
- Humidity: 42 to 69%
- Air changes (per hr): no data
- Photoperiod: 12 hr dark / 12 hr light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Pyrogen free sterile water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water):no data
- Total volume applied: 10 mL/kg bw
- Concentration in vehicle:
Expressed as active substance: 0.6, 3.0 and 6.0 mg/mL
Expressed as main ingredient: 0.45, 2.26 and 4.53 mg/mL
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test solutions daily prepared. The test substance content was checked on days 1 and 22 and found to be in an acceptable range (88-99%) when compared to the nominal concentrations.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily, 7 days a week.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 6, 30, 60 mg/kg bw.
Basis:
other: Expressed as active substance: 6, 30 and 60 mg/kg bw Expressed as main ingredient: 4.53, 22.65 and 45.30 mg/kg bw
No. of animals per sex per dose:
5 rats per sex and group (40 animals )
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: no data
- Rationale for animal assignment (if not random): no data
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: none
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: the first day of dosing and weekly.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption: collected daily per cage (5 animals) and averaged per week.

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


OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. Necropsy was performed at the end of the experiment (day 28) or immediately after killing in extremis.
Organ Weights:
organs: liver, kidneys, adrenals, testes, ovaries, thymus, spleen, brain, heart

HISTOPATHOLOGY:
– Kidney, liver and stomach as whole or as samples were preserved in formol for gross lesion observations
but tissue analysis was not processed further.


Other examinations:
None.
Statistics:
Bodyweight and organ weight data were analysed by analysis of variance and t-tests.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
see below
Mortality:
mortality observed, treatment-related
Description (incidence):
see below
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see below
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
see below
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:
effects observed, treatment-related
Description (incidence and severity):
see below
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
see below
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
- Mortality (see Table 8.9.1.1/1):
All the animals treated at 45.30 mg/kg bw (main ingredient) were killed in extremis before the end of the study. 
These animals were all killed on day 22 or before (1M at day 20, 1F at day 21). One female control was found dead on day 26, necropsy revealed no particular tissue abnormality.
- Clinic signs:
Before death, both males and females treated at 45.30 mg/kg bw (main ingredient) exhibited salivation, emaciation, rough coat condition, hypoactivity, hunched posture, noisy breathing, urogenital staining, vocalisation, ptosis and pale extremities. First occurrence of clinical signs was at the end of week 1. Four out of 5 males treated at 22.65 mg/kg bw (main ingredient) had piloerection on day 28 and one male was emaciated. Post dose salivation was seen for 3 out of 5 males and one female. These observations were done only during week 4. For the control and the low dose group (4.53 mg/kg bw, main ingredient), no particular abnormality was recorded.

BODY WEIGHT AND WEIGHT GAIN (see Table 8.9.1.1/1)
Body weight gain was severely reduced for both sexes of animals treated at 45.30 mg/kg bw (main ingredient) with weight loss observed during 
week 3. Bodyweights at the end of week 3 were 52% and 74% of controls for males and females, respectively. Body weight gain of males treated at 22.65 mg/kg bw (main ingredient) was 43% of controls for males, with weight loss for all animals seen during week 4.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study), (see Table 8.9.1.1/1):
Food consumption was reduced for males treated at 45.30 mg/kg bw (main ingredient) throughout their 3-week treatment period and for females from this group during week 3. During week 3, males ate 33% and females 42% of their respective control values.
Food consumption was reduced for males treated at 22.65 mg/kg bw (main ingredient) throughout the treatment period with overall food consumption 71% of controls. During week 4, this value was 42% of controls.
Males treated at 4.53 mg/kg bw (main ingredient) had slightly reduced food consumption (10%) during week 1 only.


ORGAN WEIGHTS (see Table 8.9.1.1/2)
In males treated at 45.30 mg/kg bw (main ingredient), absolute weight of every investigated organ has a tendency to be lower than control, except adrenals which seems unchanged.
Relative organ weights were increased for brain, heart, gonads, adrenals and kidneys, and decreased for thymus and spleen. Females seem
less affected regarding absolute weight differences with control with higher relative weight for brain, liver, adrenals and kidneys, and lower relative weight for thymus only.
Animals treated at 22.65 mg/kg bw (main ingredient) which are statistically analysed results, showed reduction of absolute weight of liver, both for males and females, heart, gonads and kidneys only in males.
The ratio between organ weight and body weight decreases for brain, gonads and kidneys for males and only for females' live.
No variation of low group organ weight was detected compared with control.

GROSS PATHOLOGY (see Table 8.9.1.1/3)
One male and one female treated at 45.30 mg/kg bw (main ingredient) had thickening of the mucosa of the stomach. The liver had pale areas and a mottled appearance for 1 male and 2 females of this group. The spleen was pale or small for 2 males and 1 female of this group and large adrenals were observed for 2 females. For animals form other experimental groups no abnormalities were recorded.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
4.53 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male
Basis for effect level:
other: clinical signs, body weight loss, reduced food consumption, reduced liver weight
Dose descriptor:
LOAEL
Effect level:
22.65 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male
Basis for effect level:
other: body weight loss, reduced food consumption, reduced liver weight

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 8.9.1.1/1: Mortality, bodyweight changes and food consumption variations

Endpoint

Controls

Low dose

Medium dose

High dose

weeks after start of treatment

1

2

3

4

1

2

3

4

1

2

3

4

1

2

3

4

Males

Mortality

(animals)

-

-

-

-

-

-

-

-

-

-

-

-

-

-

5/5

ad

Bodyweight

(g)

197

248

296

333

184

236

278

316

183

221

235*

221**

163**

167**

154**

ad

Bodyweight gain1

(g)

-

-

-

-

=

=

=

=

=

=

dec

dec

dec

dec

dec

ad

Food consumption

(g/animal/week)

175

184

186

179

157

175

179

165

158

154

128

76

131

92

61

ad

Females

Mortality

(animals)

-

-

-

1/5

-

-

-

-

-

-

-

-

-

-

5/5

ad

Bodyweight

(g)

155

176

193

204

152

167

182

193

154

174

190

197

158

171

143*

ad

Bodyweight gain1

(g)

-

-

-

-

=

=

=

=

=

=

=

=

=

=

dec

ad

Food consumption

(g/animal/week)

122

127

137

107

113

115

125

115

118

126

136

113

136

117

57

ad

ad: all dead

1: Comparison with control

* : p < 0.05, significantly different from control

** : p < 0.01, significantly different from control

dec: decrease

Significantly different from control: bold arrows (d: p <0.01)

Tendency: light arrows (d: more than 30% of difference with control)

=: no statistical significant difference with control, or tendency lower than 10%.

Table 8.9.1.1/2: Comparison of absolute weight (g) and body weight related weight (%) of main organs between controls and treated groups


Organs

Parameters

Low dose

Medium dose

High dose1

Males

Females

Males

Females

Males

Females

Brain

Absolute weight

=

=

=

=

Dec.

=

Relative weight

=

=

DEC.

=

INC.

INC.

Thymus

Absolute weight

=

=

dec.

=

DEC.

DEC.

Relative weight

=

=

=

=

DEC.

DEC.

Heart

Absolute weight

=

=

DEC.

=

DEC.

DEC.

Relative weight

=

=

=

=

inc.

=

Spleen

Absolute weight

=

=

DEC.

=

DEC.

DEC.

Relative weight

=

=

=

=

Dec.

=

Liver

Absolute weight

=

=

DEC.

dec.

DEC.

=

Relative weight

=

=

=

DEC.

=

INC.

Gonads

Absolute weight

=

=

=

=

DEC.

DEC.

Relative weight

=

=

DEC.

=

INC.

=

Adrenals

Absolute weight

=

=

=

=

=

INC.

Relative weight

=

=

=

=

INC.

INC.

Kidneys

Absolute weight

=

=

DEC.

=

Dec.

=

Relative weight

=

=

dec.

=

INC.

INC.

 

1: Data collected the day of the death, mainly on day 22.

Dec.: decrease

Inc.: increase

Significantly different from control: bold arrows (dec.: 0.01< p <0.05; DEC.: p <0.01)

Tendency: light arrows (Dec., inc. :10 to 30% of difference with control; DEC., INC. : more than 30% of difference with control)

=: no statistical significant difference with control, or tendency lower than 10%.

Table 8.9.1.1/3: Pathological observations

 

Control

Low dose

Medium dose

High dose

Clinical signs

nab

nab

Emaciation (1M)

Post dose salivation (3M/1F)

Piloerection (4M)

Hypoactivity (4M/4F)

Emaciation (4M/4F)

Urogenital staining (3M/5F)

Ptosis (1M)

Post dose salivation (5M/3F)

Noisy breathing (2F)

Necropsy findings

nab

nab

nab

Thickening of the mucosa of the stomach (1M/1F)

Liver with pale areas and mottled appearance (1M/2F)

Pale or small spleen (2M/1F)

Large adrenals (2F)

M: males; F: females

nab: no abnormalities detected

Applicant's summary and conclusion

Conclusions:
Under the test conditions of this study, the NOAEL for this study is 6 mg/kg bw/d (expressed as active substance) or 4.53 mg/kg bw/d (expressed as main ingredient) and the LOAEL is 30 mg/kg bw/day (expressed as active substance) or 22.65 mg/kg bw/day (expressed as main ingredient).
Executive summary:

Five Crl:CD(SD) BR (VAF plus) rats/ sex/ dose were given test substance at 6, 30 and 60 mg/kg bw/d, expressed as active substance, or 4.53, 22.65 and 45.30 mg/kg bw/d, expressed as main ingredient, by gavage for 28 days. The method used was similar to the OECD Guidelines 407 with some limitations (no histopathology, haematology, biochemistry and sensory reactivity observations) and in compliance with GLP.

At 4.53 mg/kg bw/d (main ingredient), only a slight reduction in food consumption was observed during the first week of the study. It was not considered to be toxicologically relevant. Marked reduction in bodyweight and food consumption were observed at dose levels of 22.65 and 45.30 mg/kg bw/d (main ingredient). Clinic signs were observed for the majority part of males and females dosed with 45.30 mg/kg bw/d (main ingredient). These signs were mainly post dosage hypoactivity, emaciation, salivation and urogenital staining for both sexes. Males from the medium dose group showed mainly post dose salivation and piloerection. Variation of absolute and/or relative weight of organs were recorded down to the medium dose level (22.65 mg/kg bw/d, main ingredient), mainly on liver and kidney, concerning mostly males. Organ pathologies were observed only in animals treated at 45.30 mg/kg bw/d (main ingredient), both for males and females, with among others liver stomach affections.

Based on these effects, the NOAEL for this study is 6 mg/kg bw/d (expressed as active substance) or 4.53 mg/kg bw/d (expressed as main ingredient) and the LOAEL is 30 mg/kg bw/d (expressed as active substance) or 22.65 mg/kg bw/d (expressed as main ingredient).