Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Toxicity to reproduction: Screening study, subacute (43-57 days), oral: gavage, Wistar rat m/f, 12 sex/group, 100, 300 and 1000 mg/kg bw/day: NOAEL = 1000 mg/kg bw/d, no toxicologically significant effects on reproduction observed (read-across from PPS, OECD 422, GLP)

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
2013-03-05 to 2013-10-18
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Klimisch 1 source record, but performed on read-across substance
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH

According to ECHA’s guidance document on information requirements and chemical safety assessment Chapter R.6 „QSARs and grouping of chemicals”, there are two techniques for grouping chemicals known when reading across to cover data gaps, i.e., category approach and analogue approach [ECHA, 2008].
A chemical category is a group of chemicals whose physico-chemical and human health and/or environmental toxicological properties and/or environmental fate properties are likely to be similar or follow a regular pattern as a result of structural similarity (or other similarity characteristic). The term analogue approach is used when the grouping is based on a very limited number of chemicals, where trends in properties are not apparent. Categories of chemicals are selected based on the hypothesis that the properties of a series of chemicals with common structural features will show coherent trends in their physico-chemical properties, and more importantly, in their toxicological (human health / ecotoxicity) effects or environmental fate properties [ECHA, 2008].
As set out in the guidance document, a chemical category is a group of chemicals whose physico-chemical and human health and/or environmental toxicological properties and/or environmental fate properties are likely to be similar or follow a regular pattern as a result of structural similarity. The similarities may be based on the following:
- common functional group(s) (e.g. aldehyde, epoxide, ester, specific metal ion);
- common constituents or chemical classes, e.g., similar carbon range numbers;
- an incremental and constant change across the category (e.g. a chain-length category), often observed in physico-chemical properties, e.g. boiling point range;
- the likelihood of common precursors and/or breakdown products, via physical or biological processes, which result in structurally similar chemicals (e.g. the metabolic pathway approach of examining related chemicals such as acid/ester/salt) [ECHA, 2008].

It is aimed to combine similarity patterns in order to cover data gaps for PPSOH. One rational for the analogue approach is the high structural similarity between the source and the target substance. 3-pyridinium-1-ylpropane-1-sulfonate (PPS) (source) and 1-(2-hydroxy-3-sulphonatopropyl)pyridinium, inner salt (PPSOH) (target) are structurally identical except an additional hydroxyl group on position 2 of the propyl moiety of the target substance. Despite the fact that a hydroxyl group may alter the toxicological or toxicokinetic behaviour of a substance, this effect is considered minor as there are three common groups in the molecules which are considered more relevant for their toxicological behaviour, i.e. the sulfo-group, the propyl moiety and the pyridine. Due to the similarities in structure, similar physico-chemical properties of the substances are to be expected, which would result in a similar toxicokinetic behaviour and most likely also in very similar toxicodynamic and toxicological behaviour. Second, the target substance is not only a metabolite of the source chemical, resulting from CYP450 metabolization (ToxTree estimation, Ideaconsult Ltd (2004-2013). Estimation of Toxic Hazard – A decision Tree approach, version 2.6.6, http://toxtree.sourceforge.net/), but they also share common metabolites, as shown from additional modelling of the source chemical metabolites (see respective table in the attachment).
Further, both substances show similar (eco-)toxicological properties in the endpoints for which data for both substances is available, which is considered proof of the suitability of the analogue approach, i.e. cross-reading from PPS to PPSOH.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

Source Chemical: 3-pyridinium-1-ylpropane-1-sulfonate / Pyridinium, 1-(3-sulfopropyl)-, hydroxide, inner salt / CAS 15471-17-7 / EC 239-491-3 (PPS), SMILES [O-]S(=O)(=O)CCC[n+]1ccccc1, MW 201.2428, C8H11NO3S

Target Chemical: Pyridinium, 1-(2-hydroxy-3sulfopropyl)-, hydroxide, inner salt / 2-hydroxy-3-pyridinium-1-ylpropane-1-sulfonate / CAS 3918-73-8 / EC 223-485-2 (PPSOH) SMILES OC(C[n+]1ccccc1)CS(=O)(=O)[O-], MW 217.2422, C8H11NO4S

Both substances do not contain impurities to an extent which is expected to alter the outcome of the experimental results or read-across approach.

3. ANALOGUE APPROACH JUSTIFICATION
Comparing the actually available information on the substances with regard to their physico-chemical properties, the minor influence of the additional hydroxyl group of the target chemical becomes obvious. All relevant information on similar metabolites can be retrieved from the respective table, in brief, the target substance is not only a metabolite of the source chemical, resulting from CYP450 metabolization, but they also share common metabolites, as shown from additional modelling of the source chemical metabolites. Considering the non-metabolized source and target chemicals only, the molecular weight only differs in the weight of a hydroxyl group and is hence in the same range, i.e. 201.24 g/mol and 217.24 g/mol, indicating per se the potential for absorption.
Both substances are solids which melt under decomposition at rather high temperatures, i.e. ≥ 245°C and have hence a negligible vapour pressure. Both compounds are very soluble in water, and their logPow is in a negative range.

In general, absorption of a chemical is possible, if the substance crosses biological membranes. In case where no transport mechanisms are involved, this process requires a substance to be soluble, both in lipid and in water, and is also dependent on its molecular weight (substances with molecular weights below 500 are favourable for absorption). Relevant for the endpoint acute toxicity dermal and skin sensitisation is the absorption resp. retention in the skin. In order to cross the skin, a compound must first penetrate into the stratum corneum and may subsequently reach the epidermis, the dermis and the vascular network. The stratum corneum provides its greatest barrier function against hydrophilic compounds, whereas the epidermis is most resistant to penetration by highly lipophilic compounds. Substances with a molecular weight below 100 are favourable for penetration through the skin and substances above 500 are normally not able to penetrate. The substance must be sufficiently soluble in water to partition from the stratum corneum into the epidermis. Therefore if the water solubility is below 1 mg/L, dermal uptake is likely to be low. Additionally logPow values between 1 and 4 favour dermal absorption. In the case of both the target and source chemical, due to their high water solubility and very low logPow, their absorption is very likely to be hindered in the stratum corneum. Nevertheless, once reaching the epidermis, i.a. due to their common small size, their absorption is favoured.
Besides the common physico-chemical and toxicokinetic properties, they exhibit a similar toxicological behaviour. Both substances are relatively non-toxic, with oral LD50 values >5000 mg/kg bw, and are non-irritating the skin and eyes.
Hence, due to the above-mentioned similarities of the source and target chemical, with regard to their structure, functional groups, toxicokinetic and toxicological behaviour, it can be reasonably concluded that a similar behaviour of the target chemical regarding its acute dermal toxicity and skin-sensitizing properties compared to the source chemical can be expected.
As indicated by studies on gene mutations in bacteria (both substances), chromosome aberrations in mammalian cells (PPS) and gene mutations in mammalian cells (PPSOH), both substances are not genotoxic. It can hence be reasonable concluded that a positive result in a chromosome mutation test on PPSOH can be excluded and read-across is justified, an underestimation of the actual hazard for genotoxic insults is unlikely. Further, as both substances are not acutely toxic, i.e. oral LD50 values are >5000 mg/kg, due to their physico-chemical properties a relevant accumulation in the body can be neglected, and no systemic or reprotoxic effects at all were noted in the OECD 422 study on PPS at the limit dose of 1000 mg/kg, the target chemical PPSOH does not need to be regarded as harmful upon repeated exposure or reproductive toxicant, too.

Besides the common physico-chemical and toxicokinetic properties, they exhibit a similar ecotoxicological behaviour. Both substances are relatively non-toxic towards aquatic invertebrates, both 48h EC50 values and even NOECs were above the limit value for classification, the EC50(48h) was even shown to be > 1000 mg/l for PPSOH. PPS showed results of LC50 (96h) > 1000 mg/L and NOEC (96h) > 1000 mg/L in the trout in an acute fish toxicity study acc. OECD 203. The EC50(72h) in algae in a study acc. OECD 201 is also above 100 mg/l, allowing in summary the conclusion that acute toxicity testing in fish would also not indicate any hazardous properties of PPSOH, so the assumption of a similar ecotoxicity profile and so read-across from PPS is also justified here.
In consequence, a similar behaviour can be expected in microorganisms. PPS is non-toxic to microorganisms, in a OECD 209 no toxicity was observed at a concentration of 1000 mg/l, so the following values were obtained for activated sludge: EC50(3h) > 1000 mg/L, NOEC(3h) = 1000 mg/L. This allows the conclusion that the substance is relatively non-toxic towards microorganisms.

Hence, due to the above-mentioned similarities of the source and target chemical, with regard to their structure, functional groups, common metabolites, toxicokinetic and ecotoxicological behaviour, it can be reasonably concluded that a similar behaviour of the target chemical regarding its ecotoxicological and toxicological properties compared to the source chemical can be expected. In summary, the target chemical PPSOH needs to be regarded as relatively non-toxic.


4. DATA MATRIX
The following table shows the available data relevant to justify the read-across from the source to the target chemical for several endpoints in order to omit testing for animal welfare:

Endpoint Source: PPS Target: PPSOH
Molecular weight 201.24 g/mol 217.24 g/mol
Physical state solid solid
Partition coefficient logPow < -2.78 at 21.5°C logPow < -2
Water solubility 240.5 g/L at 25°C (EpiSuite estimation) 1280 g/l at 23°C
Biodegradation 86 % degradation after 28 days Not readily biodegradable: no degradation observed (DOC) (OECD 301E)
readily biodegradable
Hydrolysis Not expected to undergo hydrolysis Hydrolysis can be excluded
Short-term toxicity to fish LC50 (96h) > 1000 mg/L, n/a
NOEC (96h) > 1000 mg/L (trout, OECD 203)
Short-term toxicity to aquatic invertebrates 24&48h NOEC ≥ 100 mg/L EC50(48h) > 1000 mg/l
24&48h EC50 > 100 mg/L (OECD 202) NOEC(48h) = 1000 mg/l (OECD 202)
Short-term toxicity to aquatic algae n/a EC50(72h) > 100 mg/l (OECD 201)
Toxicity to microorganisms EC50(3h) > 1000 mg/L, n/a
NOEC(3h) = 1000 mg/L (activated sludge, OECD 209)
MIC = 0.12 g/mL (Pseudomonas putida)
Acute toxicity oral LD50 > 5000 mg/kg (rat, OECD 401) LD50 > 5000 mg/kg (rat, OECD 423))
Acute toxicity dermal LD50 > 2000 mg/kg (rat, OECD 402) n/a
Skin irritation Not irritating (in vivo, rabbit) not corrosive (OECD 431, EpiDerm)
Eye irritation Not irritating (in vivo, rabbit) moderately irritant (HET-CAM, GLP)
Skin sensitization Not sensitizing (GPMT, OECD 406) n/a
Gene mutation in bacteria Negative ± S9 (OECD 471) negative ± S9 (OECD 471)
Chromosome aberration in mammalian cells Negative ± S9 (OECD 487) n/a
Gene mutation in mammalian cells n/a negative ± S9 (OECD 490)
Repeated dose toxicity NOAEL ≥ 1000 mg/kg (rat, OECD 422) n/a
Toxicity to reproduction NOAEL ≥ 1000 mg/kg (rat, OECD 422) n/a
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted 22 March 1996
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Han™:RccHan™:WIST.
- Source: Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK.
- Age at study initiation: 12 weeks.
- Weight at study initiation: 295 to 356 g (males), 188 to 217 g (females).
- Fasting period before study: no.
- Housing: Initially, all animals were housed in groups of four in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
The animals were housed in a single air-conditioned room within the testing facility.
- Diet (e.g. ad libitum): ad libitum (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK).
- Water (e.g. ad libitum): ad libitum.
- Acclimation period: 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2013-04-10 To: 2013-06-05
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
distilled
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test item was prepared at the appropriate concentrations as a suspension in Distilled water. The stability and homogeneity of the test item formulations were determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. Results show the formulations to be stable for at least twenty two days. Formulations were therefore prepared fortnightly and stored at approximately 4 °C in the dark.

VEHICLE
- Concentration in vehicle: 0, 20, 60 and 200 mg/mL for dose levels of 0, 100, 300 and 1000 mg/kg bw, respectively.
- Amount of vehicle (if gavage): 5 mL/kg bw. The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.
- Lot/batch no. (if required):
- Purity: distilled water
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 14 days
- Proof of pregnancy: [vaginal plug/ vaginal smear] referred to as [day 0] of pregnancy
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of oestrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and lactation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the test item formulation were taken and analysed for concentration of 1-(3- sulphonatopropyl)pyridinium at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. The methods used for analysis of formulations was HPLC/UV. The results indicate that the prepared formulations were within ± 6% of the nominal concentration.
Duration of treatment / exposure:
up to 8 weeks ((including a two week pre-pairing phase, pairing, gestation and early lactation for females).
Frequency of treatment:
once daily
Details on study schedule:
Chronological Sequence of Study
i. Groups of twelve male and twelve female animals were treated daily at the appropriate
dose level throughout the study (except for females during parturition where applicable).
The first day of dosing was designated as Day 1 of the study.
ii. Prior to the start of treatment and once weekly thereafter, all animals were observed for
signs of functional/behavioural toxicity.
iii. On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a
maximum of fourteen days.
iv. Following evidence of mating (designated as Day 0 post coitum) the males were returned
to their original cages and females were transferred to individual cages.
v. On completion of the pairing phase (during Week 6), five selected males per dose group
were evaluated for functional/sensory responses to various stimuli.
vi. Pregnant females were allowed to give birth and maintain their offspring until Day 5 post
partum. Litter size, offspring weight and sex, surface righting and clinical signs were
also recorded during this period.
vii. At Day 4 post partum, five selected females per dose group were evaluated for
functional/sensory responses to various stimuli.
viii. Blood samples were taken from five males from each dose group for haematological and
blood chemical assessments on Day 42. The male dose groups were killed and examined
macroscopically on Day 43.
ix. Blood samples were taken from five randomly selected females from each dose group for
haematological and blood chemical assessment on Day 4 post partum. At Day 5 post
partum, all females and surviving offspring were killed and examined macroscopically.
Any female which did not show positive evidence of mating or produce a pregnancy was
also killed and examined macroscopically.
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were chosen based on the results of previous toxicity work (Harlan Laboratories Ltd., Project Number 41300261).
- Rationale for animal assignment: The animals were randomly allocated to treatment groups using a stratified body weight randomisation procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomised.
Positive control:
None.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, soon after dosing, and one hour and five hours after dosing during the working week. Animals were observed immediately before dosing, soon after dosing and
one hour after dosing at weekends and public holidays (except for females during parturition where applicable). All observations were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioural toxicity. Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed: Gait, Hyper/Hypothermia, Tremors, Skin colour, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behaviour, Urination, Salivation Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until mating was evident. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum. Body weights were also recorded at terminal kill.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20.
For females with live litters, food consumption was recorded on Days 1 and 4 post partum.

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: Yes
Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated for females during gestation and lactation.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
Water intake was observed daily by visual inspection of water bottles for any overt changes.

OTHER:
HAEMATOLOGY
CLINICAL CHEMISTRY
NEUROBEHAVIOURAL EXAMINATION: functional perfomance tests (motor activity, forelimb/hindlimb grip strength, sensor reactivity, see section 7.5.1.)
- Pregnancy and Parturition
Each pregnant female was observed at approximately 08:30, 12:30 and 16:30 hours and around the period of expected parturition. Observations were carried out at approximately 0830 and 1230 hours at weekends and public holidays. The following was recorded for each female:
i. Date of pairing
ii. Date of mating
iii. Date and time of observed start of parturition
iv. Date and time of observed completion of parturition
Oestrous cyclicity (parental animals):
A vaginal smear was prepared for each female and the stage of oestrus or the presence of sperm was recorded.
Sperm parameters (parental animals):
Parameters examined in [P] male parental generations:
[testis weight, epididymis weight, other:]
Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell-or stage-specificity of testicular findings was noted.
Litter observations:
PARAMETERS EXAMINED
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.
For each litter the following was recorded:
i. Number of offspring born
ii. Number of offspring alive recorded daily and reported on Days 1 and 4 post partum
iii. Sex of offspring on Days 1 and 4 post partum
iv. Clinical condition of offspring from birth to Day 5 post partum
v. Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated
retrospectively from this data)

GROSS EXAMINATION OF DEAD PUPS:
[yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.]
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: Adult males were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 43.
- Maternal animals: Adult females were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 5 post partum. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 25 post coitum. Any female which failed to mate was terminated on Day 57.

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]
For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964). The corpora lutea were also counted.
All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
Samples of the tissues mentioned in table 1 were removed from five selected males and five selected females from each dose group and preserved in buffered 10% formalin.
The following tissues were preserved from all remaining animals: Coagulating gland, Epididymides, Gross lesions, Mammary gland, Ovaries, Pituitary, Prostate, Seminal vesicles, Testes, Uterus/Cervix, Vagina.
The following organs were dissected free from fat and weighed before fixation from five selected males and five selected females from each dose group: Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Pituitary (post fixation), Prostate, Seminal vesicles, Spleen, Testes, Thymus, Thyroid (weighed post-fixation with Parathyroid), Uterus (weighed with Cervix). The following tissues were weighed from all remaining animals: Epididymides, Ovaries, Pituitary (post fixation), Prostate, Seminal vesicles, Testes, Uterus (weighed with Cervix).
Postmortem examinations (offspring):
SACRIFICE
Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone.
All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.] All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
Statistics:
See "Any other information on materials and methods incl.tables".
Reproductive indices:
Mating Performance and Fertility:
The following parameters were calculated from the individual data during the mating period of
the parental generation:
i. Pre-coital Interval
Calculated as the time elapsing between initial pairing and the observation of positive
evidence of mating.
ii. Fertility Indices
For each group the following were calculated:
Mating Index (%) = (Number of animals mated/Number of animals paired) x 100
Pregnancy Index (%) = (Number of pregnant females/Number of animalsmated) x 100
Gestation and Parturition Data
The following parameters were calculated from individual data during the gestation and
parturition period of the parental generation:
i. Gestation Length
Calculated as the number of days of gestation including the day for observation of mating
and the start of parturition.
ii. Parturition Index
The following was calculated for each group:
Parturition Index (%) = (Number of females delivering live offspring/Number of pregnant females) x 100
Offspring viability indices:
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 5 of age).
i. Implantation Losses (%)
Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter as follows:
% pre–implantation loss = (Number of corpora lutea - Number of implantation sites / Number of corpora lutea) x 100
% post–implantation loss = (Number of implantation sites - Total number of offspring born /Number of implantation sites) x 100
ii. Live Birth and Viability Indices
The following indices were calculated for each litter as follows:
Live Birth Index (%) = (Number of offspring alive on Day 1/ Number of offspring born) x 100
Viability Index (%) = (Number of offspring alive on Day 4 / Number of offspring alive on Day 1) x 100
iii. Sex Ratio (% males)
Sex ratio was calculated for each litter value on Days 1 and 4 post partum, using the following formula:
(Number of male offspring /Total number of offspring) x 100
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
not specified
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no treatment-related deaths.
One female treated with 100 mg/kg bw/day died during the bleeding procedure on Day 4 post partum. This death was considered procedure related and therefore is of no toxicological significance.
There were no toxicologically significant clinical signs detected in treated animals. One male treated with 300 mg/kg bw/day had generalised scab formation between Days 19 and 23. Observations of this nature are commonly observed in group housed animals and is considered not to be of toxicological significance.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
There were no toxicologically significant effects detected in body weight development.
Males treated with 1000 and 300 mg/kg bw/day showed a statistically significant increase in body weight gain during Weeks 2 and 4 (p<0.05). Males treated with 100 mg/kg bw/day also showed a statistically significant increase in body weight gain during Week 4 (p<0.05). An increase in body weight gain is considered not to represent an adverse effect of treatment therefore the intergroup differences are of no toxicological importance.

No adverse effect on food consumption or food efficiency was detected in treated animals when compared to control animals.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
One control female failed to show any positive signs of mating and was non pregnant. One female treated with 300 mg/kg bw/day and one female treated with 100 mg/kg bw/day did not achieve pregnancy following evidence of mating. No histopathological correlates were evident in the female reproductive organs however, the male partners both showed tubular atrophy in the testes and intracellular debris and reduced sperm in the epididymides which was considered to be the cause of the non pregnancies. In the absence of any similar infertility effects detected at 1000 mg/kg bw/day the intergroup differences were considered not to be related to test item toxicity.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There were no treatment-related effects on mating performance.
No treatment-related effects on fertility were detected for treated animals, when compared to controls.

There were no differences in gestation lengths. The distribution for treated females was comparable to controls. The gestation lengths were between 22 and 23½ days.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No toxicologically significant effects were detected in the organ weights measured.
Males treated with 1000 and 300 mg/kg bw/day showed a statistically significant reduction in thyroid weight both absolute and relative to terminal body weight. The majority of individual values were within normal range for rats of the strain and age used and in the absence of any histology correlates the intergroup differences were considered not to be of toxicological importance.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No toxicologically significant macroscopic abnormalities were detected.
One male treated with 100 mg/kg bw/day had small testes and one male treated with 300 mg/kg bw/day also had small and flaccid testes and small epididymides. Microscopic examinations revealed tubular atrophy in the testes and intratubular cellular debris and reduced sperm in the
epididymides. In the absence of a similar effect at 1000 mg/kg bw/day the intergroup differences were considered not to be of toxicological importance. One female treated with 1000 mg/kg bw/day had dark kidneys at necropsy. In the absence of any histology correlates the intergroup difference was considered not to be of toxicological importance. One control male had a small and flaccid left testis and a small left epididymis. A further control male had reddened lungs. In the absence of treatment these were considered to be incidental findings.

HISTOPATHOLOGY (PARENTAL ANIMALS)
There were no treatment related microscopic abnormalities detected.
The findings recorded were within the range of normal background lesions which may be recorded in rats of the strain and age used.

OTHER FINDINGS (PARENTAL ANIMALS)
Behavioural assessment, functional performance tests, sensory reactivity, haematology and clinical chemistry parameters were comparable to those of controls.
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No toxicologically significant findings were observed in the treated animals.
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive effects
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects on reproductive performance were observed.
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOEL
Remarks:
systemic and reproductive toxicity
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects at all observed
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
In total eleven females from the control, 100, and 300 mg/kg bw/day dose groups and all females from the 1000 mg/kg bw/day dose group gave birth to a live litter and successfully reared young to Day 5 of age. The following assessment of litter response is based on all litters reared to termination on Day 5 of lactation/age.

VIABILITY (OFFSPRING)
No significant differences were detected for corpora lutea, implantation counts, implantation losses, litter size or litter viability for treated animals when compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.

CLINICAL SIGNS (OFFSPRING)
No obvious clinical signs of toxicity were detected for offspring from treated females when compared to controls. The incidental clinical signs detected throughout the control and treated groups, consisting of small size, cold, pale, missing tail and no apparent anus, no milk in stomach, found dead or missing, were considered to be low incidence findings observed in offspring in studies of this type and were considered unrelated to test item toxicity.
Litters from females treated with 100 mg/kg bw/day showed a statistically significant reduction in surface righting reflex (p<0.05). The level of statistical significance was minimal and in the absence of a similar effect in the high dose group the intergroup difference was considered to be incidental and of no toxicological importance.

BODY WEIGHT (OFFSPRING)
There were no toxicologically significant effects detected.
Statistical analysis of the litter or offspring weights data did not reveal any significant intergroup differences

SEXUAL MATURATION (OFFSPRING)
There were no intergroup differences in sex ratio (percentage male offspring) for litters from treated groups compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.

GROSS PATHOLOGY (OFFSPRING)

No treatment-related macroscopic abnormalities were detected for interim death or terminal kill offspring. The incidental findings observed were those occasionally observed in reproductive studies of this type and were considered to be unrelated to toxicity of the test item.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects at all were noted
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects were noted at all
Key result
Reproductive effects observed:
no
Conclusions:
The oral administration of 1-(3-sulphonatopropyl)pyridinium to rats by gavage, at dose levels of 100, 300 and 1000 mg/kg bw/day, did not result in any toxicologically significant effects. The "No Observed Adverse Effect Level" (NOAEL) for systemic toxicity was therefore considered to be 1000 mg/kg bw/day.
The "No Observed Effect Level" (NOEL) for reproductive toxicity was considered to be 1000 mg/kg bw/day.
Executive summary:

Introduction

The study was designed to investigate the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring development) and is designed to be compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” (adopted 22 March 1996).

This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 1000 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Distilled water).

Clinical signs, behavioural assessments, body weight change and food and water consumption were monitored during the study.

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

Extensive functional observations were performed on five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 4 post partum. Haematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group.

Adult males were terminated on Day 43, followed by the termination of all females and offspring on Day 5 post partum. Any female which did not produce a pregnancy was terminated on or after Day 25 post coitum. Any female which did not show positive evidence of mating and did not produce a pregnancy was terminated on Day 57. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Results

Adult Responses:

Mortality

There were no treatment-related deaths.

Clinical Observations

No toxicologically significant clinical observations were detected.

Behavioural Assessment

There were no treatment-related changes in the behavioural parameters measured.

Functional Performance Tests

There were no treatment-related changes in functional performance.

Sensory Reactivity Assessments

There were no treatment-related changes in sensory reactivity.

Body Weight

There were no toxicologically significant effects in body weight development.

Food Consumption

No adverse effect on food consumption or food efficiency was detected in treated animals.

Water Consumption

No adverse effect on water consumption was detected.

Reproductive Performance

Mating:

There were no treatment-related effects on mating for treated animals.

Fertility:

There were no treatment-related effects in conception rates for treated animals.

Gestation Lengths:

There were no differences in gestation lengths. The distribution for treated females was comparable to controls.

Litter Responses:

Offspring Litter Size, Sex Ratio and Viability:

Of the litters born, litter size at birth and subsequently on Day 1 and 4 post partum was comparable to controls. Sex ratio and surface righting were also comparable to controls.

Offspring Growth and Development

Offspring bodyweight gain and litter weights at birth and subsequently on Day 1 and 4 post partum were comparable to controls. No clinically observable signs of toxicity were detected for offspring from all treatment groups.

Laboratory Investigations

Haematology:

There were no toxicologically significant effects detected in the haematological parameters examined.

Blood Chemistry:

There were no toxicologically significant effects detected in the blood chemical parameters examined.

Pathology

Necropsy

No toxicologically significant macroscopic abnormalities were detected.

Organ Weights

There were no toxicologically significant effects detected in the organ weights measured.

Histopathology

There were no treatment related microscopic abnormalities detected.

Conclusion

The oral administration of 1-(3-sulphonatopropyl)pyridinium to rats by gavage, at dose levels of 100, 300 and 1000 mg/kg bw/day, did not result in any toxicologically significant effects. The “No Observed Adverse Effect Level” (NOAEL) for systemic toxicity was therefore considered to be 1000 mg/kg bw/day. The “No Observed Effect Level” (NOEL) for reproductive toxicity was considered to be 1000 mg/kg bw/day.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Study was conducted on a suitable read-across substance acc. OECD TG 422 under GLP. No toxicologically relevant effects on reproduction were noted at all up to the limit dose. Hence, the tonnage-driven data requirements under REACH are fully met, and the database is of high quality.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information

No effects on offspring development were noted in the available OECD 422 screening study on rats.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

Based on the lack of any observed adverse effects and the absence of any other test item related effects, it is rather impossible to hypothesize a concrete mode of action.

There were no adverse effects noted with regard to systemic toxicity on the parental generation in the OECD 422 screening study on a suitable read-across substance.

With regard to reproductive performance and offspring development, There were no treatment-related effects on mating for treated animals or in conception rates for treated animals. There were no differences in gestation lengths. The distribution for treated females was comparable to controls.

Regarding Litter responses, of the litters born, litter size at birth and subsequently on Day 1 and 4 post partum was comparable to controls. Sex ratio and surface righting were also comparable to controls. Offspring bodyweight gain and litter weights at birth and subsequently on Day 1 and 4 post partum were comparable to controls. No clinically observable signs of toxicity were detected for offspring from all treatment groups.

The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was hence considered to be 1000 mg/kg bw/day.

No definitive human relevance framework can be described due to the lack of any other effects securing any postulation, and no conclusion on biological plausibility can be drawn.

Despite the fact that no mode of action analysis can be performed, no data gap was identified here. The tonnage-driven data requirements under REACH were fully met, and the lack of relevance of the observed effects does also not indicate any high hazard for humans and so does not trigger any further examinations.

Justification for classification or non-classification

On the basis of the results of the reproduction/developmental toxicity screening test of a suitable read-across substance to the registered one, the no observed adverse effect level (NOAEL) of the test item was determined. The oral administration of the test item to rats by gavage, at dose levels of 100, 300 and 1000 mg/kg bw/day, did not result in any toxicologically significant effects. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore considered to be 1000 mg/kg bw/day. The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 1000 mg/kg bw/day. According to Regulation 1272/2008, Table 3.7.1(a) Hazard categories for reproductive toxicants, a substance must be considered as reproductive toxicant under the following conditions: Suspected human reproductive toxicant: Substances are classified in Category 2 for reproductive toxicity when there is some evidence from humans or experimental animals, possibly supplemented with other information, of an adverse effect on sexual function and fertility, or on development, and where the evidence is not sufficiently convincing to place the substance in Category 1. If deficiencies in the study make the quality of evidence less convincing, Category 2 could be the more appropriate classification. Such effects shall have been observed in the absence of other toxic effects, or if occurring together with other toxic effects the adverse effect on reproduction is considered not to be a secondary non-specific consequence of the other toxic effects. No effects on fertility were noted up to the limit dose of 1000 mg/kg, and no systemic toxicity was noted. Consequently the criteria for classification as reproductive toxicant (Cat. 2) are not met, the substance does not need to be classified according to Regulation 1272/2008.